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BIOLOGY
LBMIARY
A LABORATORY GUIDE IN ELEMENTARY
BACTERIOLOGY
A LABORATORY GUIDE
IN
Elementary Bacteriology
BY
WILLIAM DODGE FROST. PH. D
\\
Associate Professor of Bacteriology, University of Wisconsin
FIFTH REVISED EDITION
got*
THE MACMILLAN COMPANY
LONDOV : MACMILLAN & CO., LTD.
1918
Ml Rights Rfitried
111?
COPYRIGHT, 1901, 1902 AND 1904,
BY WILLIAM DODGE FROST.
Set up and clectrotyped. Published October, 1904. Reprinted
September, 1907; July, 1909; April, 1911; July, 1912; July, 1913;
February, 1916 ; January, 1918.
"MortoooB
Berwick & Smith Co., Norwood, Mass., U.S.A.
PREFACE TO THE FOUETH EDITION
In this edition minor changes have been made. A few of the
experiments have been rewritten; some of the old methods have
been replaced by later, and it is hoped better, methods.
The general plan of the book remains the same. Its object, as
heretofore, is to give adequate directions for the performance of
certain fundamental exercises in bacteriology. In attempting this
two considerations have been kept in mind, first, that in a rapidly
developing subject it is important that the directions for the
various exercises be worded so as to lend themselves readily to
changes which become desirable from time to time. With this
end in view the directions have been divided where possible into
a general and a special part. The general directions contain the
essential part of the exercise which does not permit of any con-
siderable variation, while the special directions embrace such
features as are most subject to modification, as for instance the
particular organism to be used, the kind of medium, the incuba-
tion temperature, etc. Desirable changes here are easily indi-
cated when the exercise is assigned ; second, that each experiment
should be complete in itself. Thus some of the experiments can
be performed in a few moments, while others require several days
for their completion. No attempt has been made to group them
into lessons. The order of the experiments is believed to be a
logical one, but may be readily adapted to meet the needs under
varying conditions.
The various bacteria are studied in groups. This arrangement
is in keeping with recent tendencies, and it is hoped that it will
impress the student with the similarity between closely related
forms, and also emphasize certain minute but important differ-
ences.
The system of classification adopted is that suggested by
Migula and is the one most widely accepted.
The nomenclature used is determined by rules generally adopt-
ed by systematists. To those who still prefer the old names, the
synonyms will be found useful.
(v)
vi PREFACE
The charts of the various organisms furnish a most satisfactory
means for recording the observations made during the study of a
germ and are especially convenient for reference.
Blank pages have been left for notes and drawing with the
idea that notes in permanent form are the only ones of value to
the student in subsequent years.
References have been made to the leading text-books and occa-
sionally to original sources. It is expected that the student will
make constant use of these references.
My acknowledgments are due to my colleagues, Professor C. A.
Fuller and Miss Vermillion Armstrong.
W. D. FROST.
Madison, Wis., January, 1911.
TABLE OF CONTENTS.
List of Apparatus X
Laboratory Eules XI
List of Keferences XII
PAET I.— GENERAL BACTEEIOLOGY
CHAPTER I. MORPHOLOGY AND ELEMENTARY TECHNIQUE.
EXERCISE PAGE
1. Cleaning Glassware 2
2. Plugging Flasks and
Tubes 4
3. Sterilization of Glassware 4
4. Preparation of Bouillon... 6'
5. Filling Test-tubes and
Flasks with Culture Me-
dia 10
6. Sterilization of Culture
Media 10
7. Preparation of Gelatin. . . 14
8. Preparation of Agar 16
9. Preparation of Potatoes.. 18
10. Preparation of Water-
blanks 18
11. Care of Culture Media 18
12. Platinum Needles 20
13. Test-tube Cultures 22
14. Incubation of Cultures. ... 24
15. Study of Test-tube Cul-
tures 26
16. Cleaning Slides and Cover-
glasses 26
17. Preparation of Staining
Solution 28
18. Simple Cover-glass Prep-
arations.. . 30
EXERCISE • PAGE
19. Use of Microscope 34
20. Drawing Bacteria 36
21. Hanging-drop Prepara-
tion 38
22. Microscopical Study of
Form Types 40
23. Study of Cell Grouping. . . 42
24. Study of Involution Forms 46
25. Study of Endospores 46
26. Flagella Stain 48
27. Capsule Stain 50
28. Stain for Metachromatic
Granules 50
29. Morphology of Yeasts and
Moulds Compared with
Bacteria 50
30. Gelatin Plate Cultures 52
31. Agar Plate Cultures 56
32. Koll Cultures 56
33. Study of Plate Cultures... 58
34. Use of Decolorizing
Agents 60
35. Gram's Stain 60
36. Tubercle Stain (Gabbett) 62
CHAPTER II. PHYSIOLOGY OF BACTERIA.
EXERCISE PAGE
37. Preparation of Special
Media 64
38. Effect of Keaction of Me-
dia on Growth 66
39. Effect of Concentration of
Media on Growth . . 66
EXERCISE PAGE
40. Effect of Temperature
Variations on Bate of
Growth 68
41. Determination of Thermal
Death Point.. 68
(VII)
viii
TABLE OF CONTENTS
CHAPTER II. PHYSIOLOGY OF BACTERIA. — Continued
42.
43.
44.
45.
46.
47.
48.
49.
50.
Comparative Efficiency of
Dry and Moist Heat 70
Effect of Desiccation..... 70
Effect of Chemicals on
Bacteria 72
Kelation to Oxygen 72
Effect of Direct Sunlight.. 72
Detection of Gas 74
Quantitative Analysis of
Gas 74
Petection of Acids and Al-
kalies 76
Quantitative Determina-
tion of Acids. . 76
EXERCISE PAGE
51. Detection of Nitrites in
Cultures 76
52. Detection of Ammonia... 78
53. Detection of Sulphuretted
Hydrogen . . 78
54. Detection of Indol 78
55. Determination of Chemical
Enzyms in Cultures. ... 80
56. Variation in Enzym Pro-
duction 80
57. Variation in Color Produc-
tion. . , 80
CHAPTER III. TAXONOMY.
Points to be Observed in the
Study of Bacteria 82
Classification of Bacteria (Mi-
gula) 89
PAGE
Bacteria Arranged in Classes
and Groups 91
CHAPTER IV. SYSTEMATIC STUDY OF REPRESENTATIVE NON PATHOGENIC
BACTERIA.
EXERCISE PAGE
58. Preparation of Special
Media 94
59. Saprophilic Class 95
60. Chromogenic Class 103
EXERCISE PAGE
62. Separation of Bacterial
Coloring Matter 108
63. Zymogenic Class 109
64. Saprogenic Class 113
65. Phosphorescent Class 121
61. Variety of Pigments 107
CHAPTER V. BACTERIOLOGICAL ANALYSIS.
EXERCISE PAGE
66. Comparative Analysis of
Air 126
67. Quantitative Determina-
tion of Number of Bac-
teria in Air 126
Water Analysis 128
Estimation of Number of
Bacteria in Soil.. . 132
68.
69.
EXERCISE
70.
PAGE
Quantitative Analysis of
Milk 132
71. Efficiency of Pasteuriza-
tion 132
72. Testing Antiseptic Action
of Chemicals 134
73. Testing Disinfecting Ac-
tion of Chemicals 136
PAKT II.— MEDICAL BACTEEIOLOGY.
CHAPTER VI. PATHOGENIC AEROBES.
EXERCISE PAGE
74. Preparation of Culture
Media 138
75. Erysipelas Group 141
EXERCISE PAGE
76. Pus Coccus Group 145
77. Malta Fever Group 153
78. Diplococcus Group 157
TABLE OF CONTENTS
ix
CHAPTER VI. PATHOGENIC AEROBES. — Continued.
EXERCISE
79.
80.
81.
82.
83.
84.
85.
86.
Sarcina Group
Anthrax Group
Friedlander Group . .
Swine Plague Group.
Glanders Group
Diphtheria Group. . .
Pneumonia Group...
Influenza Group
PAGE EXERCISE
163
167
171
183
191
199
207
211
89.
90.
91.
92.
93.
Tubercle Group 213
Colon Group 219
Hog Cholera Group 227
Typhoid Group 235
Pseudomonas Group 247
Cholera Group 251
Streptothrix Group 263
CHAPTER VII. PATHOGENIC ANAEROBES
EXERCISE PAGE
94. Emphysema Group 273
95. Oedema Group 277
EXERCISE
96. Tetanus -Group.
PAGE
, 289
CHAPTER VIII. ANIMAL INOCULATION AND STAINING OF BACTERIA IN TISSUE.
EXERCISE PAGE
97. Animal Inoculation 294
98. Preparation of Tissue for
Examination. . . . 310
EXERCISE PAGE
99. Staining Sections. 312
CHAPTER IX. BACTERIOLOGICAL DIAGNOSIS.
EXORCISE PAGE
100. Examination of Buccal
Secretion 320
101. Examination of Sputum.. 326
Examination of Blood 330
Examination of Feces 338
Examination of Urine . . . 346
102.
103.
104.
PAGE
EXERCISE
105. Examination of Transu-
dates and Exudates. . . . 346—
106. Diagnosis of Rabies 354
107. Examination of Material
from Human Autopsies 356
CHAPTER X. DETECTION OF PATHOGENIC BACTERIA IN WATER AND MILK
SUPPLIES.
EXERCISE PAGE
108. Examination of Water for
Pathogenic Bacteria. . . . 358
109. Examination of Milk for
PAGE
Pathogenic Bacteria 360
APPENDIX A.
A Key to the Indentification of the Common Pathogenic and a few of the
Well Known Saprophytic Bacteria 361
APPENDIX B.
Charts for the study of additional bacteria 365
APPENDIX C.
Tables 389
Index . . 391
LIST OF APPARATUS
This list comprises the apparatus which is to be under the
exclusive control of the student and does not include the general
laboratory outfit, such as sterilizers, incubators, microscopes, gen-
eral chemical supplies, etc.
A.
50 (% oz.) cover-glasses, 18 mm. (%
in.) square and 0.17 mm. thick
(No. 2).
50 glass slides.
100 labels, 2 cm. square.
12 cm. platinum wire (No. 27).
1 pair cover-glass forceps (Cornet
or Stewart).
1 pair fine pointed forceps.
2 slide boxes for 50 slides.
1 hanging-drop slide.
1 towel.
1 yard of muslin.
B.
1 flask, 1000 cc.
4 flasks, 400 cc.
1 flask, 250 cc.
1 flask, 100 cc.
200 test-tubes (15x120 mm.).
15 Petri dishes (10 cm.).
6 fermentation tubes.
6 glass tumblers or tin cups.
3 small wire baskets.
2 glass rods for platinum needles.
3 pipettes, 1 cc.
1 brass tube to hold pipettes (25
X 250 mm.).
8 stain bottles with pipettes, in
block.
3 sheets of filter paper.
3 sheets of lens paper.
1 test-tube brush.
1 glass funnel, 12 cm.
1 glass funnel, 5 cm.
2 stirring rods.
1 pipette, 5 cc.
1 thermometer, 0-100° C.
10 cm. rubber tubing, 1 cm. dia.
See Fig. 1.
1 Mohr stopcock.
1 potato knife.
1 Bunsen burner with tubing.
1 piece of wire gauze.
1 rice cooker.
1 graduated cylinder, 300 cc.
I graduated cylinder, 100 cc.
1 graduated cylinder, 25 cc.
1 evaporating dish, 10 cm.
1 disinfecting jar.
1 copper cup.
1 ring stand with clamp.
LABORATORY RULES
I. Before beginning an exercise read over the directions and look up
some of the references. Keep notes of everything done and the conclusions
reached on the right hand pages in this Guide. Make drawings wherever
they will be of value. Outline with pencil and fill in with India ink. The
Laboratory Guide should be kept in the laboratory.
II. All possible cleanliness should be observed in the care of apparatus,
desk, etc.
III. After working with the pathogenic bacteria the tables should be
washed with corrosive sublimate and the hands disinfected by washing in
the sublimate solution (or a germicidal soap) and then in soap and water.
IV. Solid material, culture media and corrosive sublimate should not
be put in the sink but in crocks provided for the purpose. Burnt matches,
pieces of paper, etc., should also be put in the crocks and not on the floor.
V. When using the steam sterilizer see that there is enough water pres-
ent before lighting the gas and do not leave the laboratory until the gas has
been turned off.
VI. Food should not be eaten in the laboratory and lead pencils or labels
should not be moistened with the tongue.
VII. All cultures of bacteria should be labeled with the name of the
organism, the name of the student and the date.
VIII. The platinum needles used in making cultures should be sterilized
shortly before and immediately after use, and before they are laid down.
When the needles are covered with infectious material they should be held
at the side of the flame until dry before being sterilized; this will avoid the
danger of scattering this material about the laboratory.
IX. Discarded cultures should be covered with corrosive sublimate and
placed in a proper receptacle, and under no condition should they be left
lying about the laboratory. Pipettes which have been used to handle infec-
tious material should be placed in a glass cylinder containing a disinfectant,
or potassium bichromate and sulphuric acid.
X. If infectious matter should by accident come in contact with the
hands, or be dropped on the table or floor, corrosive sublimate (1:1000) should
be immediately applied.
(XI)
LIST OF TEXTS AND REFERENCE WORKS WITH
ABBREVIATIONS USED
A. — Abbott: Principles of Bacteriology. Lea Bros. & Co., Philadelphia, 5th
Edit., 1899.
A. 2. — Abbott : Hygiene of Transmissable Diseases. Saunders & Co., Philadelphia,
2nd Edit., 1903.
B. — Bowhill : Manual of Bacteriological Technique. Oliver & Boyd, London,
2nd Edit., 1902.
C. — Chester: A Manual of Determinative Bacteriology. The Macmillan Co.,
New York, 1901.
Cn. — Conn : Agricultural Bacteriology. Blakiston's Son & Co., Philadelphia,
1901.
Cn. 2— Conn : Bacteria in Milk. Blakiston's Son & Co., 1903.
Co. — Connell : A Laboratory Guide in Practical Bacteriology. Author, King-
ston, Ontario, 1899.
Cu. — Curtis : Essentials of Practical Bacteriology. Longmans, Green & Co.,
New York, 1900.
E. — Emery : Handbook of Bacteriological Diagnosis. Blakiston's Son & Co.,
Philadelphia, 1902.
Ey. — Eyre : Bacteriological Technique. Saunders & Co., Philadelphia, 1903.
P. — Fischer : Structure and Functions of Bacteria. Clarendon Press, New
York, 1900.
Fl. — Fluegge : Die Mikro-organismen. F. C. W. Vogel, Leipzig, 1896.
Fr. — Frankland : Micro-organisms of Water. Longmans, Green & Co., New
York, 1894.
G. — Gage : The Microscope. Comstock Pub. Co., Ithaca, N. Y., 8th Edit., 1901.
Go. — Gorham : Laboratory Course in Bacteriology. W. B. Saunders & Co.,
Philadelphia, 1901.
H. — Hewlett : Manual of Bacteriology. Blakiston's Son & Co., Philadelphia,
2nd Edit., 1902.
Ho. — Horrocks : Introduction to the Bacteriological Examination of Water.
Blakiston's Son & Co., Philadelphia, 1902.
J. H. — Jordan's Translation of Hueppe : Principles of Bacteriology. Open Court
fc Pub. Co., Chicago, 1899.
v. J. — v. Jaksch : Clinical Diagnosis. Charles Griffin & Co., London, 4th Edit..
1899.
K. — Kloecker: Fermentation Micro-organisms. Longmans, Green & Co., New
York, 1903.
K. & D. — Kanthack & Drysdale : Practical Bacteriology. The Macmillan Co., New
York, 1895.
K. & W. — Kolle & Wassermann : Handbuch der Pathogenen Mikro-organismen, I., II.
and III., and atlas. Gustav Fischer, Jena, 1903.
L. — Lafar : Technical Mycology, Vol. I. Lippincott Co., Philadelphia, 1898.
Vol. II., Part I., 1903.
L. & K. — Levy & Klemperer : Clinical Bacteriology. Saunders & Co., Philadelphia,
1900.
L. & N. — Lehmann & Neumann : Atlas and Essentials of Bacteriology. W. B. Saun-
ders & Co., Philadelphia, 1901.
M. — Moore : Laboratory Directions for Beginners in Bacteriology. Ginn & Co.,
New York, 1900.
(XII)
TEXTS AND REFERENCE WORKS xiii
Mig. — Migula : System der Bakterien. Gustav Fischer, Jena, 1900.
M. & B. — Muir & Ritchie : Manual of Bacteriology. The Macmillan Co., New York,
3rd Edit., edited by Harris, 1903.
M. & W.— Mallory & Wright : Pathological Technique. W. B. Saunders & Co., Phila-
delphia, 2nd Edit., 1903.
McF. — McFarland : Text-Book of Pathogenic Bacteria. W. B. Saunders & Co.,
Philadelphia, 4th Edit., 1903.
N. — Novy : Laboratory Work in Bacteriology. Geo. Wahr, Ann Arbor, Mich.,
2nd Edit., 1899.
Ne. — Newman : Bacteria. Putnam, New York, 2nd Edit., 1903.
P. — Park : Bacteriology in Medicine and Surgery. Lea Bros. & Co., Philadel-
phia, 1899.
P. B. C. — Proceedings of the Bacteriological Committee from Jour. Amer. Pub. Health
Assn., Vol. XXII.
P. & M. — Peamain & Moor : Applied Bacteriology. Baillere, Tindall & Cox, London,
2nd Edit.
P. & W. — Prescott & Winslow : Elements of Water Bacteriology. Wiley & Sons, 1904.
R. — Roger : Infectious Diseases. Lea Bros. & Co., Philadelphia, 1903.
S. — Sternberg : Manual of Bacteriology. Wood & Co., New York, 1893.
S. 2. — Sternberg : Immunity. Putnam & Sons, New York, 1903.
Si.— Simon : Clinical Diagnosis. Lea Bros. & Co., Philadelphia, 3rd Edit., 1897.
W. — Woodhead : Bacteria and their Products. Charles Scribner & Sons, New
York, 1892.
Wm. — Williams : Manual of Bacteriology. Blakiston's Son & Co., Philadelphia,
3rd Edit., 1904.
PART I
GENERAL BACTERIOLOGY
PART I- GENERAL BACTERIOLOGY
CHAPTER I
MORPHOLOGY AND ELEMENTARY
TECHNIQUE
EXERCISE 1. CLEANING GLASSWARE.
GENERAL DIRECTIONS. All glassware to contain culture media
must be thoroughly cleaned. New glassware should be washed in
hot soap-suds (a test-tube brush will be needed for the test-tubes),
rinsed in tap water and then placed for a few minutes in water to
which about \% of hydrochloric acid has been added to remove
free alkali frequently present on new glass, and then thoroughly
rinsed in running water. It is then allowed to drain. Test-tubes
and flasks are best dried by placing them on a drain board spe-
cially prepared, or standing them mouth down in a box with a
cloth bottom or in a wire basket.
Glassware containing media (discarded cultures, etc.,) is best
cleaned by first standing in water for some hours, or by being
steamed and pouring out the material while in a liquid condition
and then cleaning as above with the exception of the use of the
hydrochloric acid.
REFERENCES. A. 126 ; H. 44 ; P. 223.
SPECIAL DIRECTIONS. Read Rule I. Clean as directed above,
all flasks, test-tubes, fermentation tubes and Petri dishes in your
possession.
GENERAL BACTERIOLOGY
EXERCISE 2. PLUGGING FLASKS AND TUBES.
GENERAL DIRECTIONS. When the flasks, test-tubes and fer-
mentation tubes are thoroughly dry they are to be plugged with
cotton. The cotton for this purpose should be non-absorbent and
of the best quality, i. e., as free from foreign matter as possible.
The plugs should be sufficiently loose to permit the interchange of
gases and at the same time tight enough to support the weight of
the vessel and its contents, otherwise they are apt to be pulled out
in handling the vessels. The cotton should be rolled into a cylin-
der of the proper diameter and long enough to extend into the
mouth about 2y2 cm. (1 in.) and project sufficiently to protect the
lips from dust. The plug should be pushed in straight and not
twisted; the surface next to the glass must be perfectly smooth,
presenting no creases for the entrance of dust.
REFERENCES. A. 127; H. 44; M. & R. 49; McF. 164; P. 223.
SPECIAL DIRECTIONS. Plug all test-tubes, flasks and fermenta-
tion tubes in your possession.
EXERCISE 3. STERILIZATION OF GLASSWARE.
GENERAL DIRECTIONS. The glassware thus prepared is ready
for sterilization, which process is accomplished in an apparatus
called the hot air sterilizer. This is a sheet iron or copper box with
a double wall which permits of rapid
heating. The apparatus should be so ar-
ranged that a temperature of 150° C. can
be quickly reached and readily main-
tained. In such a sterilizer all glassware
L to be used for the reception of culture
media, such as flasks, test-tubes, Petri
dishes, etc., is submitted to a temperature
of 140-150° C. for 1 hour, or until the
cotton plugs are slightly browned; this
change being due to the incipient char-
ring of the cotton. The test-tubes are
placed erect in square baskets made of
galvanized iron wire. When the air in
the sterilizer has cooled to about 40° C.
the glassware can be taken out and stored ready for use. The Petri
dishes are not to be opened until used for culture purposes.
REFERENCES. A. 75 and 127 ; H. 36 ; M. & R. 29 ; McF. 164 ;
P. 223.
;x
Fig. 1. Hot Air Sterilizer,
(Muir & Ritchie).
6
GENERAL BACTERIOLOGY
SPECIAL DIRECTIONS. All glassware prepared in 1. is to be
sterilized for one hour at 150° C. The small pipettes should be
placed in brass tubes, provided for the purpose, and also sterilized.
EXERCISE 4. PREPARATION OF BOUILLON.
GENERAL DIRECTIONS. Any one of the three methods (A, B
or C) may be used. They are arranged in order of preference, but
method C is the most convenient, and hence most used.
A.
a. From 500 grams (1% Ibs.) of lean
beef remove the fat and connective tis-
sue and mince (or use Hamburg steak).
b. Add 1 liter of distilled water and
after shaking thoroughly set in ice
chest for 12 to 24 hours.
c. Squeeze through a cloth and add
enough distilled water to filtrate to
make 1 liter and place in vessel to cook.
C.
a. Weigh out
three grams of
beef extract
(such as Lie-
big's).
6. Add 1 liter
of distilled wa-
ter.
c. Place in
vessel for cook-
ing.
B.
a. Ditto.
b. Add 1 liter of
distilled water.
c. Place in ves-
sel for cooking,
then cook for ys
hour at about 70°
C., filter through
paper and make
up to 1 liter.
d. Add to any of the above solutions: \% (10 gms.) peptone
(Witte) and %% (5 gms.) common salt (NaCl), then weigh solu-
tion, with vessel, so that the water which is subsequently driven
off in cooking can be accurately replaced.
Cooking may be done either in a flask which is heated in a water-
bath or sterilizer, double-walled boiler, or rice-cooker. In case a rice-
cooker is used a 50% solution of calcium chloride should be placed
in the outer vessel instead of water as by this means the contents
of the inner vessel can be brought to a rapid ebullition, something
impossible by the use of water alone.
e. Heat, not above 60° C., until ingredients are in solution,
then restore the water lost by evaporation.
/. Neutralize. This is a very important step and calls for great
care. Of the following methods, A is more accurate and should
be employed for special or research work. For ordinary routine
work B may be employed.
GENERAL BACTERIOLOGY
A.
1.) Titrate as follows: Pipette off 5
cc. of the fluid into a 10 cm. evaporating
dish, add 45 cc. of distilled water, boil
for three minutes, add 1 cc. of phen-
olphthalein (0.5% substance in 50% al-
cohol), and then run in carefully, drop
by drop, from a burette a twentieth
normal1 solution of sodium hydroxide
(^NaOH) until the solution turns a
faint pink color. Treat two other sam-
ples in the same way. If the amount of
NaOH required is approximately the
same in each case the average can be
taken as the amount necessary to neu-
tralize 5 cc. Calculate the amount nec-
essary to neutralize the whole (1000—15
cc.). Since this amount would dilute
the medium too much, a stronger solu-
tion (normal) is used, hence,
Use a normal1 solution of so-
dium hydroxide (£NaOH). Add
to the hot solution a few cc. at a
time at first, later a few drops,
stirring thoroughly with a glass
rod. After each addition, test
by placing a drop of the solution
by means of the glass rod on a
strip of phenolphthalein paper.
(Prepared by dipping filter pa-
per in a solution.) The addition
should continue until the test pa-
per is turned a faint pink color.
2.) Neutralize by adding -gVtli of the volume calculated above
of a normal solution of sodium hydroxide. Test the accuracy of
the work at this point by the addition of a few drops of phenol-
phthalein to a cc. or so of the medium. If a faint pinkish tint is
not obtained, titration and neutralization must be repeated.
g. Boil for 5 minutes and restore weight.
h. Test reaction and adjust if necessary.
i. Add 0.5 to 1.5% of a normal hydrochloric acid. The amount
of acid to be added varies with the purpose for which the medium
is to be used, e. g., in water analysis + 1.0 (acid) is preferable,
with the pathogenic bacteria a smaller amount of acid (J-0.5)
more nearly meets requirements.
j. Heat until precipitate appears flaky and then filter through
moistened filter paper. (For method of folding see Abbott p. 96).
The filtrate (bouillon) should be of a light straw color, per-
fectly clear, and should not give a precipitate on boiling.
REFERENCES. A. 94; H. 45; M. & B. 35; McF. 180; P. 212;
P. B. C. 18-24.
SPECIAL DIRECTIONS. Prepare 1 liter of bouillon according to
method C. Secure and put to soak meat for 7. See Rule IV.
1 Normal solutions are prepared so that one liter at 16° C. shall contain
the hydrogen equivalent of the active reagent weighed in grams (Sutton).
For present purposes a 4% solution of sodium hydrate is sufficiently accurate.
10
GENERAL BACTERIOLOGY
EXERCISE 5.
FILLING TEST-TUBES AND FLASKS WITH CULTURE
MEDIA.
GENERAL DIRECTIONS. In filling tubes be careful not to allow
the media to touch the neck of the vessel as this will cause the
cotton to stick to the glass when the plugs are removed. Place
the culture fluid to be tubed in a funnel arranged with a delivery
tube and stopcock (Fig. 2), from which it can be
run into sterile vessels. Test-tubes should contain _ _
6-10 cc. of medium (about 3 cm. deep). Flasks
are to be filled about three-fourths full.
SPECIAL DIRECTIONS. Fill 15 test-tubes and
preserve remainder of bouillon in flasks.
EXERCISE 6. STERILIZATION OF CULTURE
MEDIA.
EXPLANATORY. To sterilize culture media steam
is used almost exclusively either as streaming FIG. 2. Appara-
steam or under pressure. The unconfined steam tubes*
is applied in an apparatus known as a steam ster-
ilizer. Of the various patterns the Arnold is perhaps the most
satisfactory. It is effective, economical in the use of gas, and
does not allow the escape of large quantities of steam into the
room as a large part is condensed to be reconverted into steam.
A simple steam sterilizer is shown in
>•• Fig. 3, and for student use is very
convenient. The method of using
either form is identical. Always
have plenty of water present before
heating. Exposure is made on three
consecutive days for 20 minutes, be-
ginning to count time when the ma-
o« terial reaches the temperature of the
FIG. 3. Simple sterilizer consisting steam> which wil1 varv with different
of a galvanized iron pail with a cover substances and the volume treated.
a ana a raise bottom o.
Between successive steamings culture
media should be kept under conditions favorable to spore germi-
nation,' i. e. , at the room temperature. This method of steriliza-
tion is known as the discontinuous method or Tyndalization.
For 'the employment of steam under pressure the autoclave is
essential. The lid should contain a thermometer as well as a
12
GENERAL BACTERIOLOGY
steam gauge, safety and outlet valve. A thermo-regulator is also
desirable. The following table gives the temperature correspond-
ing to atmospheres of pressure indicated on the gauge :
TABLE OF TEMPERATURES CORRESPONDING TO STEAM-
PRESSURES.
Temperatures
Steam-Pressure
Temperatures
Steam-Pressure
F.
C.
Lbs.
F.
C.
Lbs.
212°
100°
0
251°
121.5°
15
228°
109°
5
260°
126.5°
20
240°
115.5°
10
287°
141.5°
40
This table is true only when all of the air in the
apparatus is replaced by steam, and hence the steam
must be allowed to escape freely before the outlet
valve is closed. A single exposure of 20 minutes at
a temperature of 120° C. (one additional atmos-
phere) is sufficient to kill all germ life. After the
proper exposure, care must be taken not to allow the
steam to escape too rapidly, otherwise the culture
media may be forced against the plugs owing to the
unequal pressure.
GENERAL DIRECTIONS. Ordinary media may be
sterilized by either method. In case of gelatin and
sugar media the temperature should not exceed
110° C. for 15 minutes.
O O O 0 <
REFERENCES.
166; P. 218.
FIG. 4. Autoclave;
A. 59-77 ; H. 37 ; M. & E. 29 ; McF. a, safety valve; &,
blow-off pipe; c.
gauge; (M u i r &
Kitchie).
SPECIAL DIRECTIONS. Sterilize bouillon pre-
pared in 4 for 20 minutes in a steam sterilizer on three consecutive
days, or in the autoclave at 120° C. for 20 minutes. Rule V.
N. B. Some time is required to raise the temperature of the
media to that of the steam, especially if the vessels are large.
All media should be carefully examined every day for a week or
more, and if "specks" or the least cloudiness appears, the medium is
not sterile and the process of sterilization must be repeated.
14 GENERAL BACTERIOLOGY •
EXERCISE 7. PREPARATION OF GELATIN.
GENERAL DIRECTIONS.
Same as bouillon (4).
c.
d. Add \% peptpne, 0.5% salt and 10-15 %* of the best gold
label, sheet gelatin, and weigh.
e. Heat until ingredients are dissolved.
/. Neutralize.
g. Boil 5 minutes and restore weight.
h. Test reaction.
i. Boil until albumin coagulates and floats in the clear fluid. If
beef extract is used it will be necessary to first cool below 60° C.
and thoroughly stir in an egg.
j. Filter. Arrange the apparatus
shown in Fig. 5. Use absorbent cotton.
The funnel and flask should first be heated
with warm water. Usually the hot gelatin
will filter without the use of the pump. If
the pump is needed it should be started be-
fore pouring in the culture medium. This
prevents the unfiltered gelatin from pass-
ing between the cotton and glass.
Jc. Add 5.0 cc. (0.5 %) of a normal hy-
, 1 -I • • i i ,• FIG. 5. Apparatus for fllter-
arOCnloriC acid. SOlUtlOn. ing media through absorbent
7 m v /K\ cotton; a, coil of wire over
I. lUDe. (D). laid with layer of absorbent
0, .,. . ^ „ OA cotton; b, tubes for making
m. Sterilize in the Steamer for 20 connection with air pump; c,
minutes on three consecutive days or in the ^ance^/waterlnto^Isks. er
autoclave at 110° C. for 15 minutes.
REFERENCES. A. 99 ; H. 47 ; M. & R. 40 ; McF. 184 ; P. 215 ;
P. B. C. 26.
SPECIAL DIRECTIONS. Make 1 liter, using method A. Fill 30
test-tubes. Put the remainder in flasks, sterilize in steam sterilizer
or autoclave. Remember long exposure to high heat injures the
solidifying properties of gelatin.
1 The amount to be varied according to the season of the year, 10 per cent,
in winter, 12-15 per cent, in summer, but it should be remembered that differ-
ent quantities affect the appearance of the cultures.
16 GENERAL BACTERIOLOGY
EXERCISE 8. PREPARATION OF AGAR (RAVENED).
GENERAL DIRECTIONS.
Add 15 grams of agar-agar threads (finely chopped; to 500 cc.
of water and either (1) dissolve in autoclave by heating up to 120°
C., closing off gas and allowing to cool, or (2) boil until the agar-agar
is dissolved (about y2 hour) and make up loss of water by evapora-
tion. While the agar is being dissolved proceed as follows :
a. Same as in the preparation of bouillon (4 a).
fc. Add 500 cc. of distilled water.
c. Same as bouillon (4 c).
d. Add 10 gms. of peptone and 5 gins, of salt.
e. Heat until peptone is dissolved.
/. Neutralize.
g. Cool to 60° C., add agar solution and mix (in case extract is
used it will be necessary to add an egg at this point) .
h. Boil until albumin is coagulated and floats in the clear liquid
and restore weight.
i. Test reaction.
,;'. Add 0.5% normal hydrochloric acid.
~k. Filter as in case of gelatin. (7j.)
I Tube.
m. Sterilize in steam for 15 minutes on three successive days or
in autoclave for 20 minutes at 120° G.
After the last sterilization place most of
the tubes in a sloping position to harden
(Fig. 6) , these are known as agar slopes.
Those solidified in an upright position, —
frequently called "deep stick agar," »»•«• Method of sloping agrar,
are used to make plate cultures.
REFERENCES. A. 104 ; H. 47 ; M. & R. 38 ; McF. 185 ; P. 215 ;
P. B. C. 27 ; Journal of Applied Microscopy, 1898, 1 ; 106.
SPECIAL DIRECTIONS. Use meat extract, make 1 liter, fill 25
tubes and after last sterilization incline 20 of them. Place the
remainder in flasks and sterilize.
18 GENERAL BACTERIOLOGY
EXERCISE 9. PREPARATION OF POTATOES (BOLTON).
GENERAL DIRECTIONS.
a. Select a number of rather large test-tubes (150x20 mm.),
place a small wad of absorbent cotton1 in the bottom
of each (Fig. la), plug and sterilize as usual.
~b. Wash a large potato, then with a cork borer
slightly smaller than the test-tubes punch out cylinders
about 5-6 cm. long.
c. Divide these diagonally and trim to shape indi-
cated in Fig. 7 &.
d. Add a few drops of distilled water to each test-
tube and place pieces of potato in position.
e. Sterilize on three consecutive days for 30 to 45
minutes. FlG.7. Boiton's
Unless the tubes are to be used immediately, they potato tube'
should be sealed. (11.) The dark color can be prevented by im-
mersing the pieces between c and d in running water for 12-18 hours.
REFERENCES. A. 107 ; H. 49 ; M. & . 47 ; McF. 190 ; P. 216 ; P.
B. C. 28 ; S. 47.
SPECIAL DIRECTIONS. Prepare 15 test-tubes of potato, sterilize
and seal with paraffin. (11.2.)
EXERCISE 10. PREPARATION OF WATER-BLANKS.
GENERAL DIRECTIONS. Water-blanks are prepared by placing
exactly 10 cc. of a physiological salt solution (6 gms. per 1,000 cc.
water) in test-tubes and sterilizing in autoclave 15 minutes at 120°
C., or in steamer 15 minutes on three successive days.
SPECIAL DIRECTIONS. Prepare and sterilize 10 water-blanks.
EXERCISE 11. CARE OF CULTURE MEDIA.
When sterile culture media (or test-tube cultures) are to be
kept for some time they must be protected from evaporation and
stored in a dark, cool place. Evaporation may be checked to a con-
siderable extent, (1) by storing them in tin cans, e. g. quinine cans.
Care must be taken, however, that these do not become too damp in
which case the mould fungi frequently grow through the cotton
plugs; (2) flasks and test-tubes may be sealed by removing the
1 Gage recommends glass beads. A smaller cylinder of potato may also be
used instead of the cotton; in this case the tubes would be sterilized empty.
20 GENERAL BACTERIOLOGY
plugs, dipping same in melted paraffin (melting point about 50° C.)
and then replacing them; (3) by cutting off the projecting cotton
and drawing over the mouth of the vessel a rubber cap (made for the
purpose) which has been sterilized in a solution of mercuric bi-
chloride, or rubber dam, easily obtained from dentists, fastened on
with a rubber band, may also be used ; or (4) by use of a cap of tin-
foil. In this case the foil should be put on as soon as the tubes are
filled, and sterilized with the medium.
All media should be carefully examined every day for a week or
more, and if spots or the least cloudiness appears, the medium is not
sterile and the process of sterilization must be repeated.
All receptacles containing media should be labeled after steriliza-
tion. For this purpose labels can be purchased, the size used for
glass slides, or gummed paper in sheets can be cut into squares
( 2 cm. ) . The labels are to be attached to each ves-
sel 1 cm. from the lip. The name of the student,
the kind of medium and the date of preparation
should be written across the top, leaving the rest of
the label to be filled in when the medium is inocu-
lated. Rule VII.
EXERCISE 12. PLATINUM NEEDLES.
GENERAL DIRECTIONS. These are made by fusing a piece of No.
27 platinum wire (5 cm. long) into a glass rod or tube (18 cm.
f ^ long). (Fig. 8.) The dan-
ger of having the wire crack
O- c ] off when the needle is heated
FIG. 8. Platinum Needles. is lessened if a little piece
(1/2 cm.) of fusible glass is soldered on the glass rod before the
wire is melted in. Each student should have two such needles ; in
one the wire should be straight (designated " needle") and the other
bent to form a ' ' loop. ' ' This loop should be formed around a No. 10
wire. These instruments must be sterilized shortly before and im-
mediately after use by heating the wire to a glow in the gas flame.
The handle should also be passed through the flame two or three
times. Cool before using. If the habit of sterilizing is thoroughly
acquired much trouble will be avoided and possible danger prevented.
These needles will be in constant use.
REFERENCES. A. 131 ; H. 42 ; M. & R. 51 ; McF. 196 ; P. B. C. 33,
foot note.
22
GENERAL BACTERIOLOGY
EXERCISE 13. TEST-TUBE CULTUEES.
EXPLANATORY. The extreme minuteness and slight variation in
the form of different bacteria render a thorough study of them by
direct microscopic observation a difficult and well nigh impossible
task. In their study, therefore, it is necessary to depart from the
usually accepted rules that govern the determination of the life
history of other forms of life and resort to special methods. The
most successful of these are those known as culture methods. Ac-
cording to these methods the bacteria are sown on various food sub-
stances and upon these they develop forming masses easily visible
to the naked eye. The manner of their growth and the changes
which they produce in these media make it possible to detect differ-
ences which would otherwise escape attention. The most common
culture media, bouillon, gelatin, agar, and potato have already been
prepared, and others will be described as needed.
Cultures may be made either in test-tubes (streak or stab cult-
ures), or on glass plates, as plate cultures. The plate culture is
especially important and is used (a) to obtain pure cultures; and
(6) for ascertaining the character of the colonies as an aid to
their diagnosis. The tube-cultures are serviceable in giving oppor-
tunity for a further study of the characters as well as to furnish the
most convenient method of maintaining the cultures.
GENERAL DIRECTIONS. Bacteria when obtained in "pure
culture" are usually grown in test-tube cultures. To make these
a small portion of a previous culture is transferred to fresh culture
media by means of the platinum needles.
a. Stab Cultures are made in test-tubes containing solid, trans-
parent media, such as gelatin
and agar. The end of a ster-
ile needle is infected with the
material to be transferred.
The needle is then thrust into
the medium to the bottom of
the test-tube and withdrawn.
In this way the bacteria are
left along the entire length
of the needle track. For
method of holding tubes see
Fig. 9. They are held in an
inclined position to prevent
the possibility of infection ^ g Method of holding test.tubes.
from the air. «, cotton plugs.
24 GENERAL BACTERIOLOGY
b. Streak Cultures are cultures made by drawing the needle, or
better, the loop, over the surface of the medium (test-tubes with
media having sloped surfaces or plate cultures). Agar, potato and
blood serum are frequently used in this way, and occasionally
gelatin.
c. Liquid Cultures (bouillon, milk, etc.) are inoculated by trans-
ferring the desired material to them on either the needle or loop.
REFERENCES. A. 152 ; H. 58 ; M. & R. 51 ; McF. 198.
SPECIAL DIRECTIONS.
a. Make a gelatin stab, an agar streak, a potato streak, and a
bouillon culture of Bacil lus subtilis (EHRENB.) COHN (hay bacillus)
and Bacillus coli (Escn.) MIG. (colon bacillus) from agar cultures
supplied. Rule VIII.
b. Label each tube, writing the name of the organism, the date of
inoculation and your own name. Rule VII.
c. Place the gelatin in the cool chamber, and the other cultures in
the incubator at 28° C. See next Exercise.
EXERCISE 14. INCUBATION OF CULTURES.
EXPLANATORY. Most bacteria grow at ordinary temperatures
(22° C.), but their growth is usually hastened by a higher tempera-
ture (e. g. 28°-30° C.) The pathogenic, or disease-producing bac-
teria grow best at the temperature of the human body (38° C.).
All bacteriological laboratories are, therefore, supplied with appara-
tus arranged for maintaining constant temperatures, known as
thermostats or incubators.
The non-pathogenic cultures are usually kept at 28° C., while
the pathogenic ones are kept at 38° C. All gelatin cultures, how-
ever, must be kept at a temperature several degrees below the melt-
ing point of gelatin, i. e., not above 22° C. Ordinarily the tempera-
ture of the locker, especially near the floor, will be found satisfac-
tory. In a very warm room, particularly in the summer, an artifi-
cially cooled chamber will be necessary.
Test-tube cultures are stored in the various incubators in tin
cans or glass tumblers with a layer of cotton in the bottom, while
the Petri dishes are stacked in low piles.
REFERENCES. A. Ch. VIII ; H. 55 ; M. & R. 82 ; P. 231.
SPECIAL DIRECTIONS.
a. Incubate all cultures of the non-pathogenic bacteria at 28° C.,
BACILLUS STJBTILIS (EHRENB.) COHN 25
Gelatin Stab: Grown 24hours at °C.
48 hours at
=c.
6 days at <>C.
Agar Streak: Grown 24 hours at °C.
I
! I
48 hours at
6 days at °C.
Potato: Grown 24 hours at
9
•s
48 hours at °C.
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at.
26 GENERAL BACTERIOLOGY
except the gelatin. Keep these in the cool chamber. After growth
has taken place, the cultures can be taken from the incubator and
kept at the room temperature.
1}. Study and make diagrams of an incubator, a Reichert thcrmo-
regulator, a Roux thermo-regulator and Koch 's safety burner.
EXERCISE 15. STUDY OF TEST-TUBE CULTURES.
GENERAL DIRECTIONS. As soon as growth becomes visible a sys-
tematic and careful study of the cultures should be made. A de-
tailed list of the points to be noted will be found in Chapter III,
and should be consulted in writing up the descriptions. The sum-
mary below will, however, be found useful.
For bouillon cultures note : 1 ) condition of fluid, 2 ) character
of sediment, 3) presence or absence of membrane, and 4) charac-
teristic odor.
For solid cultures (agar and potato slopes), note: 1) Form of
growth, 2) size, 3) surface elevation, 4) consistency, 5) color, 6)
effect on media, and 7) characteristic odor.
For gelatin stab cultures, note : 1) Effect on media, a. non-lique-
fying, i) line of puncture, ii) surface, &. liquefying, i) shape of
liquefied area, ii) condition of fluid, iii) character of sediment, 2)
characteristic odor.
The study should be continued from day to day as long as
changes are noted. Make drawings wherever they will be of
service in elucidating the descriptions.
SPECIAL DIRECTIONS. Study, write careful descriptions and
make necessary drawings of all the cultures made. For recording
results use the table on pages 25 and 27.
EXERCISE 16. CLEANING SLIDES AND COVER GLASSES.
GENERAL DIRECTIONS. Slides can be sufficiently cleaned by
washing in water or alcohol and drying with a towel. The cover-
glasses for bacteriological work, however, must not only be freed
from visible dirt but must be rendered free from fat. One of the
best methods is the following: New cover-glasses are cleaned by
washing in water and drying from alcohol by rubbing them between
driers (two wooden blocks 20x10x2^ mm. covered with several
layers of cotton cloth or chamois skin), and then heating them on
BACILLUS COLI (Escn.) MIG.
Gelatin Stab: Grown 24 hours at.
5C.
Agar Streak: Grown 24 hours at
O i
48 hours at
6 days at oC
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at..
28 GENERAL BACTERIOLOGY
a piece of sheet iron or in hot air sterilizer for one hour at about
200° C. They are best kept in a clean Petri dish and handled
with forceps. (Novy). Old slides and covers having balsam on
them should first be dropped one by one into a cleaning solution
(potassium bichromate 60, sulphuric acid 60, water 1,000), and
boiled for one-half hour and then treated as above.
SPECIAL DIRECTIONS. Clean y2 oz. of cover-glasses and place
them in a clean Petri dish.
EXERCISE 17. PREPARATION OF STAINING SOLUTIONS.
GENERAL DIRECTIONS. The dyes most useful for staining bac-
teria are the basic anilin dyes which come in powdered or crystal-
line form. (Gruebler's dyes are standard). Those in most com-
mon use are Fuchsin, Methylen blue, Gentian violet and Bismarck
brown. They keep in powdered form, with perhaps the exception
of Methylen blue, but because of greater convenience and equally
good keeping qualities, saturated alcoholic solutions are kept in
stock. These are made by adding the dry dye to 95% alcohol to
saturation and filtering. This form can not be used for staining
bacteria. The following solutions are required to begin work with :
1. Aqueous solution of Gentian violet.
Saturated alcoholic solution of Gentian violet 2.5 cc.
Distilled water 47.5 cc.
2. Saturated aqueous solution of Bismarck brown.
3. Ziehl's carbol-fuchsin.
Saturated alcoholic solution of Fuchsin 5 cc.
Solution of carbolic acid (5%) 45 cc.
4. Loeffler's Methylen blue.
Saturated alcoholic solution of Methylen blue 15 cc.
Potassium hydrate (IrlOjOOO)1 50 cc.
5. Ehrlich's Anilin Oil Gentian violet.2
Saturated alcoholic solution of Gentian violet 6 cc.
Absolute alcohol 5 cc.
Anilin water . 50 cc.
1 This dilution can be readily made by taking 1 cc. of a 10% potassium
hydrate solution, making this up to 100 cc., then taking 5 cc. of this and
making it up to 50 cc.
2 Some prefer anilin oil made as follows : Solution A., 2 cc. of anilin oil
and 100 cc. of distilled water; Solution B., 25 cc. of filtered saturated alco-
holic solution of gentian violet ,and 75 cc. of Solution A. Mix and filter.
This stain remains good for a long time.
30 GENERAL BACTERIOLOGY
Anilin water is prepared by adding 2-3 cc. of anilin oil, drop by drop, to
50 ec. of water, thoroughly shaking and then filtering through moistened
paper until perfectly clear.
This stain should stand 24 hours and then be filtered. It does not keep
well and must not be used when more than 14 days old.
6. Gram's Iodine solution.
Iodine 1 gm.
Potassium iodide 2 gm.
Distilled water 300 cc.
7. Gabbett's Methylen blue solution.
Methylen blue (dry) 2 gms.
Sulphuric acid 25 cc.
Distilled water 75 cc.
8. Alcohol, 96%.
REFERENCES. A. 163 ; H. 85 ; M. & R. 97 ; P. 200.
SPECIAL DIRECTIONS. Prepare the
solutions of dyes from the saturated
alcoholic solutions (furnished) and
place them in 2 oz. bottles arranged
with pipettes and neatly labeled. The
bottles are conveniently kept in a
block. Fig. 10.
EXERCISE 18. SIMPLE COVER-GLASS
PREPARATION FIG. 10. Block for stain bottles.
GENERAL DIRECTIONS. Bacteria may be studied under the micro-
scope in a living condition in a hanging drop preparation (21) ;
but on account of their hyaline character, which makes the exami-
nation difficult, the student should first learn to stain them and
later make the hanging drop preparation. With a few exceptions
all bacteria can be stained by the following process : A small drop
(about the size of a pinhead) of distilled water is placed on a clean
cover-glass by means of the platinum loop. With a sterile needle a
portion of the material to be examined is secured and while the
cover-glass is held in the fingers of the left hand the bacteria on the
needle are introduced into the water, thoroughly mixed and then
spread in a thin film over as much of the surface of the cover-glass
as possible. When the bacteria are taken from fluid media a drop
of water will not be necessary. In this case use a loop. The film
is now allowed to dry. If the drop is sufficiently small this will be
a short process. It may be hastened by holding the cover-glass high
32 GENERAL BACTERIOLOGY
over the flame, but it should always be held in fingers to prevent over-
heating, which spoils the preparation.
"When the film is thoroughly dry place the cover-glass in a pair
of Cornet or Stewart forceps and "fix" the bacteria in the flame.
This is done by passing the prepa.ra.tion
through the upper portion of a gas fla
film side up. Three passages should be
made, each consuming about one second forceps. (Muir& Ritchie).
of time. The forceps are now placed on the table and the film flooded
with one of the anilin dyes. After the stain has acted for five to
ten minutes it is washed off into a waste dish with a stream of dis-
tilled water, and while the cover-glass is still wet it is placed, bac-
teria side down, on a clean glass slide, being careful to avoid air
bubbles. The surplus water is then taken up by means of a small
piece of blotting or filter paper.
The preparation is now ready for microscopical examination.
(For directions see next exercise).
The preparation can be made permanent either by allowing the
water under the cover-glass to dry before it is removed, or by floating
it off with water and afterwards drying. When dry a drop of
Canada balsam, dissolved in xylene, is placed on the cover-glass and
this is then lowered on to the slide again.
Resume.
a. Spread film,
b. Air dry,
c. Fix,
d. Stain,
e. Mount in water, / \ e. Dry,
f. Examine, 3 or v /. Mount in balsam,
g. Dry and mount in balsam. ( ) g. Examine.
"The great mistake made by beginners is to take too much
growth," (M. & K.) and too large a drop.
REFERENCES. A. 159 ; H. 80 ; M. & R. 98 ; McF. 145 ; P. 198 ;
P. B. C. 11.
SPECIAL DIRECTIONS.
a. Make cover-glass preparation from agar streak of B. subtilis
(13) staining with an aqueous solution of gentian violet for five
minutes.
34 GENERAL BACTERIOLOGY
b. Practice making cover-glass preparations by staining speci-
mens from each of your cultures. Use Loeffler's methylen blue for
the gelatin and bouillon ; aqueous solution of gentian violet for agar,
and carbol-fuchsin for potato. Examine, mount permanently and
hand to instructor for inspection.
EXERCISE 19. USE OF MICROSCOPE.
GENERAL DIRECTIONS. For bacteriological purposes a microscope
with a magnifying power of at least 500 diameters is needed. There
should be a coarse adjustment (rack and pinion) as well as a fine
micrometer screw ; and the following accessories : Two oculars, one
1 in. (25 mm.) and one 2 in. (50 mm.) ; three objectives, one 2 in.
(16 mm.), one -J- in. (4 mm.), or | in. (3.5 mm.) and one oil
immersion -fa i*1- or TS" in- (2 mm.) ; a triple nose-piece, and an
Abbe substage condenser with iris diaphragm mounting.
In the use of the microscope the following points should be noted :
a. LIGHT. The proper angle at which the mirror should be
placed is best determined by removing the ocular and so arranging
the mirror that the unobstructed light from the window covers the
whole field. The ideal light is that from a white cloud. Direct
sunlight should never be used.
~b. ABBE CONDENSER. The purpose of the condenser is to furnish
a large cone of light, and as it is corrected for parallel rays the plane
side of the mirror should always be used, except when artificial light
is employed. When highly stained objects are to be examined, the
open diaphragm should be used, but when the structural rather than
the color picture is desired, it will be necessary to diminish the light
by closing the diaphragm. When the high powers are employed,
raise the condenser as high as possible ; for low powers a lower posi-
tion will give better definition.
c. FOCUSING. Turn the proper objective in place and rack down
until the objective nearly touches the cover-glass. This should be
done while the eye is held at one side and directs the movement.
Then with the eye at the tube slowly move up with the micrometer
screw. Never rack down with the eye at the tube.
d. USE OF OIL-IMMERSION. The oil-immersion objective is indis-
pensable to the proper study of bacteria. It is constructed upon the
principle that a drop of fluid having the same refractive index as the
36 GENERAL BACTERIOLOGY
objective, prevents the dispersion of light, thus permitting the use
of lenses having a greater numerical aperture and longer working
distance for the same degree of amplification than is possible with
the dry system. In using an immersion lens, place a small drop of
immersion oil on the preparation, then carefully lower the objective
until it touches the oil drop and nearly touches the cover-glass.
Apply eye to the ocular and focus upward very slowly with fine ad-
justment until the definition is clear. At the close of the day's
work the oil must be removed from the objective and cover-glass.
This is best accomplished by wiping them with a piece of Japanese
paper made for the purpose. In case the oil should accidentally dry
on the objective, it can be removed by adding a little more oil and
allowing it to stand for a few minutes; it can then be wiped off
with paper. If this method does not succeed, the objective should
be taken to the instructor. Great care must be observed since
solvents of the oil are also solvents for the lens mountings.
KEFERENCES. See Gage ; A. 199 ; H. 118 ; M. & R. 87 ; P. 206.
SPECIAL DIRECTIONS. Examine cover-glass preparations made in
previous exercise, first with \ in. objective, and then with the oil-
immersion objective. If the specimen be satisfactory, sketch as
directed in next exercise.
EXERCISE 20. DRAWING BACTERIA
GENERAL DIRECTIONS. In drawing bacteria only a few organisms
occurring in the microscopic field should be sketched, but these should
be made of considerable size so that the exact outline may be indi-
cated. Furthermore they should be drawn to scale and individuals
selected to give range in form and size.
To measure microscopic objects an ocular micrometer is used,
and the first step will be to determine its value. Place the ocular
micrometer on the diaphragm in the ocular, use a stage micrometer
as an object and focus. The image of the scale on the stage micro-
meter will appear imposed on that of the ocular micrometer. Make
the lines of the two micrometers parallel and then make any two
lines of the stage micrometer coincide with any two on the ocular
micrometer, pulling out the draw-tube if necessary. Divide the
value of the included space or spaces on the stage micrometer by
the number of divisions on the ocular micrometer required to in-
clude them, and the quotient so obtained will give the valuation of
the ocular micrometer in fractions of the units of measure of the
stage micrometer ( Gage) . If the result be not in terms of the micron
38
GENERAL BACTERIOLOGY
it should be converted to such, as this is the unit in micrometry.
REFERENCES. G. 100-108.
SPECIAL DIRECTIONS.
a. Determine the value of the ocular micrometer and fill out
blanks in following table :
No of IMicroscope
IMake
Ocular i
n., or mm.
Objective.
Tube length.
Value of single di-
vision on scale
in fA.
%in. (16mm.)
%in. (4mm.)
Oil-immersion.
&. Measure the bacteria on the preparations made in Exercise 18
and sketch a few individuals from each.
In making drawings, represent a micron by two and one-half
millimeters on paper. This will give a magnification of 2,500 diame-
ters, represented thus : X 2,500.
EXERCISE 21. HANGING-DROP PREPARATION.
GENERAL DIRECTIONS. These are made by adding a small portion
of bacterial culture from solid media to a drop of water on a clean
cover-glass, or, in case of fluid media, by placing a small loopful of
the culture medium on the cover-glass. A hollow ground glass slide
having the rim of the cavity previously coated with vaseline, is
inverted and lowered over the cover-glass enclosing the drop. With
a careful, quick movement the preparation is now brought right
side up.
2
FIG. 12. Hanging-drop preparation, a, Hanging drop; &, Vaselin.
40 GENERAL BACTERIOLOGY
Instead of the hollow ground glass-slide an ordinary glass-slide
to which a small section of a glass or rubber tube has been cemented
can be used, and in some cases is preferable.
In examining the preparation under a microscope, focusing is a
somewhat difficult process and must be carried out with great care.
Use a narrow diaphragm. Find the edge of the drop with the low
power (•§• in. objective), adjusting slide so that edge of drop passes
through the center of the field; then turn on the high power (-J- in.
objective) and focus without moving the slide. The edge of the drop
is selected because the bacteria are here nearest the cover-glass and
hence more easily focused upon than where they are deeper in the
drop.
REFERENCES. A. 204 ; H. 114 ; M. & R. 87 ; McF. 141 ; P. 209.
SPECIAL DIRECTIONS.
a. Make a hanging-drop preparation of water containing parti-
cles of India ink or carmine in suspension. This illustrates molec-
ular or Brownian movement.
ft. Make a preparation. using straw infusion or tartar from teeth
to note variations in rate and character of vital movement.
c. Make hanging-drop preparation of B. subtilis from agar or
bouillon (13).
d. Make same preparation of B. coli (13).
In cases where vital movement is questionable, remove the cover-
glass and place a drop of formalin or chloroform in the bottom of the
cell ; replace the cover-glass, examine and note change in character
of movement, if any.
EXERCISE 22. MICROSCOPICAL STUDY OF FORM TYPES.
a. Make bouillon and agar streak cultures of the following or-
ganisms :
Micrococcus (any species).
Sarcina lutea SCHROETER.
Pseudomonas fluorescens (FLUEGGE) MIG.
Bacillus mycoides FLUEGGE.
Microspira Metschnikovi MIG. (or any vibrio).
Spirillum rubrum v. ESMARCH.
I. Incubate cultures at 28° C. for 24 hours.
42 GENERAL BACTERIOLOGY
c. Make cover-glass preparations from the agar streaks and stain
with an aqueous solution of gentian violet or with Loeffler 's methylen
blue.
d. Examine with the oil-immersion objective, and write the
names of the organisms in their proper places in the table below :
Shape of
organism.
Eelative
size.
Name.
Sketch.
Spherical.
Medium.
Small.
Elongated.
Large.
Small.
Spiral.
Short.
Long.
e. Make sketches of each organism.
/. Mount all preparations in balsam and hand them to instructor
for inspection.
EXERCISE 23. STUDY OF CELL GROUPING.
IMPRESSION PREPARATIONS. The exact relation of cell to cell as
they develop in the colony can frequently be determined best by
studying a "contact preparation" which is prepared as follows :
a. Melt a gelatin tube and slope it, when solid make a streak
culture of B. mycoides and when growth has taken place dip the tube
in hot water to loosen gelatin, which is then slipped out of the tube.
6. Lower gently a clean cover-glass over the surface. Apply a
slight pressure by tapping glass. Raise cover-glass by one edge,
taking care that natural arrangements of adherent bacteria are not
disturbed.
c. Thoroughly air dry the same, then fix and stain in the ordinary
manner.
44
GENERAL BACTERIOLOGY
d. Examine the thinner layers, noticing the arrangement of cells
with reference to each other, and draw a sufficient number to illus-
trate this relationship.
HANGING-DROP PREPARATIONS.
a. Make hanging-drop preparations from bouillon cultures pre-
pared above (22) and also from those supplied.
&. Examine with oil-immersion objective and assign organisms
to their proper places, as determined by cell grouping, in the follow-
ing scheme :
Arrangement.
Form.
Name.
Sketch.
Isolated.
Spheres.
Kods.
Spirals.
Filaments.
Spheres.
Rods.
Spirals.
Plane surfaces.
Spheres.
Eegular masses.
Spheres.
Irregular masses.
Spheres.
Eods.
AGAR HANGING-DROP CULTURES (Wesbrook).
a. Melt a tube of agar and cool to 43° C.
Z>. Sterilize a cover-glass by passing it two or three times through
the flame quickly.
c. With the needle make a streak on the cover glass about 3 mm.
long of B. subtilis.
46
GENERAL BACTERIOLOGY
d. With the loop place a drop of liquid agar so as to cover up
streak.
e. Flame a hollow-ground slide and seal the cover glass to it. In-
cubate and later examine and sketch.
REFERENCES. Hill, Hanging Block, Jour. Med. Research, 1902,
2; 202.
EXEECISE 24. STUDY OF INVOLUTION FORMS.
a. Grow Bacillus subtilis (EHRENB.) MIG. in bouillon, and also
in water containing 0.1% asparagin, 10% sugar, and by means
of stained cover-glass preparations compare the individual organ-
isms in each case in regard to their form and size. The degenerated
or involution forms are more apparent by staining. Draw several
cells illustrating a variety of involution forms.
~b. Examine a culture of Bacterium diphtheriae (LOEFFLER) MIG.
on Loeffler's blood serum. Read M. & R. 5.
EXERCISE 25. STUDY OF ENDOSPORES.
a. Make cultures on peptoneless agar, or on an agar tube to which
a few drops of calcium hydrate have been added, of the following
organisms and incubate at 28° or 38° C. :
Bacillus subtilis (EHRENB.) COHN.
Bacterium anthracis (Kocn) MIG. (or Bacillus mycoides
FLUEGGE).
Bacillus amylobacter VAN TIEGHEM (or any clostridium form).
Bacillus tetani NICOLAIER (or any "drumstick" bacillus).
ft. When the cultures are 48 hours old mount films without stain-
ing, examine and fill out following table :
Size of Spore.
Position.
Name of organism.
Sketch.
Smaller than di-
ameter of mother-
cell.
Median.
Polar.
Larger than diam-
eter of mother-
cell.
Median.
Polar.
48 GENERAL BACTERIOLOGY
c. Simple stain for spores.
1. Prepare film of B. subtilis.
2. Fix by passing through flame 10 or 12 times instead of 3 times.
(This prevents the vegetative portion from taking the stain).
3. Stain 2-5 minutes in hot carbol-fuchsin.
4. Mount and examine.
d. Double stain for spores (Hauser's method).
1. Prepare a film of any of the above organisms (providing a
previous examination has shown that the spores are fully developed
and the mother-cells have not disintegrated).
2. Fix, three times through the flame.
3. Stain with hot (steaming) carbol-fuchsin for 5 minutes.
4. Cautiously decolorize with 5 per cent, acetic acid until the
pink color is nearly removed from the film.
5. Wash thoroughly in water.
6. Dry (blot).
7. Stain with Loeffler's methylen blue, 3 minutes.
8. Mount and examine. The spores should appear crimson in
blue bacilli.
KEFERENCES. A. 171 ; H. 98 ; M. & R. 106 ; McF. 154 ; P. 46 &
203 ; P. B. C. 15.
EXERCISE 26. FLAGELLA STAIN (BUKGE).
GENERAL DIRECTIONS.
a. Make an agar streak of the organism to be stained.
I). After 18 to 24 hours, by means of the platinum needle re-
move a portion of the growth (being careful to avoid the culture
medium) to a large drop of tap water on a perfectly clean cover-
glass (16). Allow this to stand 5 minutes rather than spread, as
there is less danger of breaking off the flagella.
c. Spread carefully 2 or 3 loopfuls of this drop on each of sev-
eral clean cover-glasses and dry at room temperature.
d. Fix by passing the cover-glass while it is held in the hand,
(not in the forceps, as over-heating will injure the preparation)
through the top of the flame.
e. Flood the cover-glasses thus prepared with the following solu-
tion (mordant) : Liquor ferri sesquiMoridi diluted with distilled
water 1 : 20, 1 part ; saturated aqueous solution of tannic acid, 3
parts. This mixture improves with age but should be filtered before
using. Allow to act 1 minute.
f. Wash in water and dry between filter paper.
50 GENERAL BACTERIOLOGY
g. Stain with hot carbol-fuchsin for about one minute.
h. Wash in water, dry and mount in balsam.
KEFEBENCES. A. 174 ; H. 100 ; M. & R. 107 ; McF. 156 ; P. 205.
SPECIAL DIRECTIONS. Stain B. typhosus from cultures fur-
nished, also try B. coli and B. subtilis.
EXERCISE 27. CAPSULE STAIN (WELCH).
GENERAL DIRECTIONS.
a. Spread film without the use of water,
fc. Air dry.
c. Fix.
d. Apply glacial acetic acid, and drain it off immediately. Do
not wash in water.
e. Apply Ziehl's carbol-fuchsin which is to be renewed several
times to remove acid.
/. Wash in 1 to 2% salt solution.
g. Examine in salt solution. (Balsam causes capsule to shrink).
REFERENCES. A. 170 ; H. 97 ; M. & R. 106 ; McF. 291 ; P. 203 ;
P. B. C. 13.
SPECIAL DIRECTIONS. Use pneumonic ("rusty") sputum, blood
of rabbit infected with the Bact. pneumoniae or a milk culture of a
capsule bearing organism as Bact. pneumonicum (Fried.) Mig. or
Bact. capsulatum (Stern.) Chester.
EXERCISE 28. STAIN FOR MET ACHROMATIC GRANULES (ERNST).
a. Stain a young culture of an organism such as Bact. diphtheriae
with Loeffler 's methylen blue for about 3 minutes.
b. Wash in water.
o. Treat with a saturated solution of Bismarck brown for 30 sec-
onds.
d. Wash in water, mount in water and examine, or, dry, mount
in balsam and then examine.
The granules should appear blue in a brown organism.
EXERCISE 29. MORPHOLOGY OF YEASTS AND MOULDS COMPARED
WITH BACTERIA.
a. Mount some baker's yeast (Sac char omyces cerevisiae) and
examine in an unstained condition. Compare: Size; form; struc-
ture and method of reproduction with the bacteria.
~b. In same way examine a number of common moulds, e. g.
Mucor, Penicillium and Aspergillus.
GENERAL BACTERIOLOGY
EXERCISE 30. GELATIN PLATE CULTURES.
EXPLANATORY. Plate cultures are only possible with the lique-
fiable solid media, gelatin and agar. In making them the bacteria
are mixed with the medium while it is in a fluid state and spread out
on a horizontal surface to cool. The dilution is such that the indi-
viduals are separated from each other by several millimeters. In
the solidified medium the organisms are fixed and their growths
result in the formation of "colonies." These vary in size and ap-
pearance according to the peculiarities of the organism and the age
of the culture, but are of the greatest service in the study and identi-
fication of the various species. These cultures are prepared as fol-
lows:
GENERAL DIRECTIONS. Three gelatin tubes are marked Nos. 1, 2
and 3 and melted by placing them in a water bath at a temperature
of 42° C. For this purpose a small cup of
water placed on a tripod can be used (Fig.
13). They are inoculated by introducing the
material to be studied into tube No. 1. The
quantity of this material varies. The amount
clinging to the platinum needle will be suffi-
cient if a pure culture be used, while in other
cases several loops or even drops are necessary.
The inoculated material is thoroughly mixed
with the gelatin in No. 1. This is done by
rolling the tube gently between the palms of
FIG. 13. Method of melt- the hands> instead of shaking, so as to prevent
ing gelatin. the introduction of air bubbles. With a ster-
ile loop two loopfuls of fluid gelatin are now transferred from No. 1
to No. 2, and mixed. For method of handling tubes see Fig. 14.
In like manner
three or more
loops from No. 2
are carried over
to No. 3, which
in turn is well
mixed. The con-
tents of each of
the tubes are
now poured into
PIG. 14. Another method of holding test-tubes. separate sterile
Petri dishes. The process of pouring is performed as follows : The
54
GENERAL BACTERIOLOGY
Petri dish is placed on the desk; the gelatin tube is taken in the
right hand, the cotton plug removed with the left hand ; the mouth
of the tube sterilized by naming it once or twice, and, when the glass
is cool, the gelatin is
poured into the lower half
of the dish while the cover
is slightly raised (Fig.
15), but not inverted or
T • -. ,, , i i mi FIG. 15. Method of pouring plates.
laid on the table. The
cover of the dish is then replaced, the test-tube filled with a solu-
tion of corrosive sublimate, and the cotton plug returned. The
gelatin is spread over the entire bottom of the dish by tipping it
from side to side. It is then allowed to harden by placing the dish on
the cooling apparatus, or leaving it on a horizontal surface at room
temperature. A simple, inexpensive and effective cooling apparatus
is a piece of soapstone, such as is
sold at hardware stores (Fig.
16). In winter this can be
cooled by hanging it out of
doors, at other seasons by im-
mersing it in cold water. The
three Petri dishes thus prepared
should be properly labeled and
placed under conditions where the gelatin will remain solid and yet
growth will take place. The temperature of the laboratory should
not be allowed to exceed 23° C. or gelatin cultures are in danger of
melting while under examination. Within a few days colonies will
make their appearance, in varying numbers, depending upon the
dilution used.
Inasmuch as the first plate is generally too thickly seeded to be
of much service, this gelatin tube is often replaced by a water blank,
which is treated exactly as the gelatin tube No. 1, but is not, of course,
"plated" but used simply to dilute the material.
KEFERENCES. A. 130 ; H. 65 ; M. & R. 53 ; McF. 199 ; P. 224.
SPECIAL DIRECTIONS.
a. Make three gelatin plate cultures, as directed above, and inoc-
ulate with B. subtilis, introducing a minute portion of agar culture
(13) into tube No. 1, two loops of No. 1 into No. 2, and three of No.
2 into No. 3. Label, and when the gelatin has solidified, place plates
in cool chamber (14).
FIG. 16. Soapstone used for solidifying
gelatin in Petri dishes.
56
GENERAL BACTERIOLOGY
6. Also make a "blank" plate from an uninoculated gelatin tube,
observing all precautions to prevent contamination. This will serve
as a control or check on your other plates. If any -colonies develop
on this it will indicate carelessness.
EXERCISE 31. AGAR PLATE CULTURES.
GENERAL DIRECTIONS. These are made in the same way as the
gelatin plates except that the high melting point (96° C.) of agar
makes it necessary to use boiling water to melt it. Inasmuch as the
vitality of vegetative bacteria is destroyed at a temperature much
above 42° C. it must be cooled down before it is inoculated, but as
agar solidifies at 39-40° C. it must not, therefore, be cooled below
that point. It is best to keep thg melted agar at about 45° C. for 10
minutes before it is inoculated. For this purpose a water-bath
should be so arranged that the temperature can be controlled by
means of a thermo-regulator. A cheap and
yet satisfactory arrangement is represented
in Fig. 17. Inoculate, make dilutions and
^K g pour as in case of gelatin, except that be-
_\\ — I I — I I—I R fore the agar is poured, it is well to slightly
warm the Petri dishes by placing them in
the incubator at 38° C. for a few minutes,
otherwise the agar may solidify in lumps in
the plate. In cooling, agar shrinks some-
what, and in doing so water is expressed
from the solid jelly. In the incubator this
condenses on the under side of the cover of
PIG. 17. Water-bath for cool- the Petri dish to such an extent that drops
run down on to the culture surface, thus
causing the developing superficial colonies to "run." To obviate
this the Petri dishes, when placed in the incubator, should be in-
verted
REFERENCES. A. 135 ; H. 68 ; M. & R. 57 ; N. 285 ; P. 225 ; P. B. C.
28.
SPECIAL DIRECTIONS, a. Make three agar plates of B. coli; use
one loop of bouillon culture (13) for tube No. 1 and proceed as in 30.
6. Invert and place in incubator at 28° C.
EXERCISE 32. ROLL CULTURES (ESMARCH).
GENERAL DIRECTIONS. These are essentially plate cultures in
which the medium instead of being poured out into dishes is solidi-
58
GENERAL BACTERIOLOGY
FIG. 18. Method of making Roll-cultures. (Abbott).
fied in a thin, even layer on the inner surface of the test-tubes. This
is best accomplished by means of a piece of ice placed in a dish on
a piece of cloth, by which it can be kept in the desired position
(Fig. 18). A hori-
zontal groove is melt-
ed in the ice by means
of a test-tube filled
with hot water. In
this groove the test-
tubes, inoculated as
in case of plate cul-
tures, are rapidly
whirled until the me-
dium is thoroughly
set. Both agar and
gelatin can be used,
although gelatin cannot be used successfully with those species which
liquefy this medium. In the case of agar the tubes should be placed
in a horizontal position a few hours (over night) until the medium
has become attached to the tube : afterwards they can be stored in
the usual receptacles for tube cultures.
REFERENCES. A. 137 ; H. 69 ; M. & R. 56 ; McF. 206.
SPECIAL DIRECTIONS, a. Melt a tube of gelatin and without inoc-
ulating it practice making a roll-culture as described above. Avoid
tipping the tube enough to get medium on cotton plug. Remelt and
roll again and again until the knack is acquired.
~b. Make two roll-cultures in gelatin of E. coli (13) , using a water-
blank instead of gelatin tube No. 1.
c. Make two agar cultures of B. siibtilis in same way.
d. Incubate 6. in cool chamber, and c. at 28° C.
EXERCISE 33. STUDY OF PLATE CULTURES.
MACROSCOPIC. As the colonies appear, note: a. form, &. size,
c. surface elevation, d. consistency, e. color. Both the surface and
deep colonies should be described, as they are frequently very differ-
ent. Drawings should always be made wherever they will be of
value; study should be continued as long as changes are noticed.
(See Chapter III.)
MICROSCOPIC. The colonies appearing on the plates are to be
studied under a low power of the microscope. Use a -f- in. (16 mm.)
60 GENERAL BACTERIOLOGY
objective. The Petri dishes can be inverted, and thus avoid the
danger of exposing the culture to contamination from the air except
with gelatin where liquefying organisms are present. Observe, a.
structure of colony as a whole ; &. character of margin. (See Chap-
ter III.)
SPECIAL DIRECTIONS. Study, write descriptions and make draw-
ings of all plate cultures. Use blank pages for description and
sketch of cultures.
EXERCISE 34. USE OF DECOLORIZING AGENTS.
Make three cover-glass preparations from a 24 hour old culture
of B. subtilis, staining them with an aqueous solution of gentian
violet. Mount in water and examine. While they are still under
the microscope, place at one side of the cover-glass a few drops of
one of the following solutions, and by means of a strip of filter paper
at the opposite side draw the liquid under the cover glass until all
the color is removed. In this way determine the relative value of
alcohol (95%), acetic acid (5%), and nitric acid (30%) as decolor-
izing agents.
EXERCISE 35. GRAM'S STAIN.
EXPLANATORY. This is a differential stain and one of the most
useful. Some bacteria when stained by this method exhibit a dark
violet color, others remain perfectly colorless, thus rendering pos-
sible the differentiation of bacteria which are morphologically nearly
or quite identical, and also greatly facilitating the demonstration of
certain bacteria in animal tissue. Most of the pathogenic micrococci
retain the violet stain, although there are important exceptions.
The bacilli and spirilla may or may not remain colored.
GENERAL DIRECTIONS.
a. Spread film.
b. Air dry and fix.
c. Stain with anilin-oil gentian violet 1% minutes.
d. Pour off stain and without washing.
e. Apply Gram's iodine solution (17, 6) 1% minutes.
/. Apply 96% alcohol 3 minutes, or until drippings do not stain
white filter paper.
g. Wash in water.
62 GENERAL BACTERIOLOGY
h. Mount in water and examine.
i. Dry and mount in balsam.
REFERENCES. A. 169 ; H. 89 ; M. & B. 102 ; McF. 150 ; P. 203.
SPECIAL DIRECTIONS. Stain films of young cultures of B. coli
and B. subtilis.
EXERCISE 36. TUBERCLE STAIN (GABBETT).
EXPLANATORY. All of the differential methods of staining the
tubercle bacterium depend upon the fact that this germ is very
resistant towards the ordinary stains, and, in order to be stained at
all must be treated with a dye containing a mordant and this either
allowed to remain in contact with the micro-organism several hours
or be applied hot. The latter method is the quicker and is usually
employed, although it does not give as good results. When once
stained this germ withstands the effect of decolorizing agents to
such an extent that it is possible to remove the dye from all other
objects on the cover-glass preparation (as in sputum) while it retains
its own color. The application of a second dye, of a complementary
color, readily distinguishes this germ from all others in the field. A
few other bacteria have similar staining qualities. Red is the usual
stain and blue the counter stain. Gabbett's method is one of the
simplest.
GENERAL DIRECTIONS.
a. Spread film (sputum from tuberculous patient).
fe. Air dry and fix.
c. Stain with hot carbol-fuchsin 2 minutes.
d. Wash in water.
e. Treat with Gabbett's solution % to 1 minute.
/. Wash in water and examine.
g. Dry and mount in balsam.
REFERENCES. A. 167 ; H. 244 ; M. & R. 104 ; McF. 308 ; P. 304.
SPECIAL DIRECTIONS. Stain three samples of sputa which con-
tain varying numbers of the tubercle bacteria.
64 GENERAL BACTERIOLOGY
CHAPTER II
PHYSIOLOGY OF BACTERIA
EXERCISE 37. PEEPAEATION OF SPECIAL MEDIA.
The following media will be necessary for the work outlined in
this chapter :
a. DEXTROSE BOUILLON. To ordinary bouillon add \% dextrose
(c. P.), tube and sterilize in steamer, not in autoclave, 7 test-tubes
and 2 fermentation tubes.
&. DEXTROSE GELATIN. \% dextrose (c. P.), tube and sterilize
in steamer, 6 tubes.
c. DEXTROSE AGAR. \% dextrose (c. P.), tube and sterilize in
steamer, 5 tubes.
d. LACTOSE AGAR. \% lactose (c. P.), tube and sterilize in
steamer, 2 tubes.
e. LITMUS SOLUTION. To 10 gms. of the dried material add 500
cc. of distilled water, digest in a warm place, decant clear liquid and
add a few drops of nitric acid to produce a violet color. (Button.)
Place in flasks or test-tubes and sterilize in steamer three times, 1
tube.
/. DEXTROSE-FREE BROTH. This is prepared from beef by inocu-
lating the meat infusion with an organism capable of fermenting
sugar, such as B. coli, and allowing it to stand several hours at 38° C.
(Between &. and c. Exercise 4.) The bouillon is then prepared in
the usual manner.1
Or DUNHAM'S SOLUTION.
Sodium chloride 0.5 gm. ) _. ., ., n . -,. , , «,,
.__. . f Boil until all is dissolved, filter,
Peptone (Witte) 1. gm. V .,. . , ,
' _ _ f tube and sterilize, 4 tubes.
Water 100. gms. )
g. NITRATE SOLUTION.
1 Smith: Jour. Exp. Med., 1897, 2: 543.
66 GENERAL BACTERIOLOGY
Sodium chloride 0.5 gm. ""j
Peptone (Merck) 1. " ! -r,.,, . , .,. _ . ,
A _ V Filter, tube and sterilize, 3 tubes.
Potassium nitrate 0.2 '
Water 1,000. gms. J
h. LITMUS MILK.
1) Freshly separated milk, or if this is not available, new milk
is placed in a separatory funnel in an ice chest over night to allow
the separation of the cream and the milk then drawn off.
2) Litmus solution (e. above) is then added until medium is
faintly blue.
3) Tube and sterilize in the steamer for 30-45 minutes on 3 or
4 consecutive days. During the summer months particularly very
resistant bacterial forms abound in the milk, so that it is necessary
to increase the number of applications or length of exposure. The
efficiency of the sterilizing process should be tested by placing the
milk in the incubator for several days to see if any change occurs,
2 tubes.
In addition to the above have 15 tubes of bouillon (9 to contain
exactly 10 cc. for 41. and 44.) 10 tubes of gelatin, 15 tubes of agar,
6 water-blanks and 5 potato tubes.
( If thought desirable the media required for Chapters IV. and V.
[Exercise 58], may be prepared at this time; this would then com-
plete all the media making required in Part I. )
EXERCISE 38. EFFECT OF REACTION OF MEDIA ON GROWTH.
GENERAL DIRECTIONS.
a. Melt 6 tubes of gelatin and add, under aseptic precautions, to
three of them, respectively, 0.1 cc., 0.3 cc., and 0.5 cc. of a normal
solution of hydrochloric acid, and to the other three the same
amounts of a normal sodium hydrate solution.
fc. Thoroughly mix, solidify gelatin in ice water and then inocu-
late (stab) each tube with the organism to be studied.
c. Make a control culture in a tube of neutral gelatin.
d. Incubate at 18° C. and note the effect of the chemicals on the
rate, amount and character of the growth.
KEFERENCES. L. & N. 35 ; McF. 41.
SPECIAL DIRECTIONS. Use B. subtilis and B. coli. Make sketches.
EXERCISE 39. EFFECT OF CONCENTRATION OF MEDIA ON GROWTH.
a. Pour about 2 cc. of " condensed milk" into each of two sterile
test-tubes, dilute one with five times the volume of sterile water.
68 GENERAL BACTERIOLOGY
Z>. Inoculate both with a pure culture of B. subtilis and incubate
at 28° C. Explain changes which occur.
c. Test extract of beef or syrup in the same way.
EXERCISE 40. EFFECT OF TEMPERATURE VARIATIONS ON RATE OF
GROWTH.
GENERAL DIRECTIONS.
a. Make four agar streak cultures of organism to be studied.
It. Incubate them at the following temperatures: Ice chest (7°
C.), room (20° C.), low incubator (28° C.), blood heat (38° C.).
c. By frequent observations as to luxuriance of growth, deter-
mine the optimum temperature of growth for each.
KEFERENCES. F. 73 ; L. & N. 44 ; McF. 44.
SPECIAL DIRECTIONS. Use a mesophilic bacterium as B. coli and
a psychrophilic organism as Ps. violacea.
EXERCISE 41. DETERMINATION OF THERMAL DEATH POINT.
GENERAL DIRECTIONS.
a. Make a bouillon culture of the organism to be tested.
~b. 48 hours later heat a large water-bath to 45° C. Place in
this, in close proximity to a thermometer, 5 test-tubes (16 mm. in
diam.) containing exactly 10 cc. of standard bouillon. (Reaction
+ 1.5.)
c. After 15 minutes exposure at this temperature remove the
cotton plug from one of the tubes, inoculate the broth with three
loopfuls (standard size, 12) of the 48 hour old culture (a.), and
carefully mix by slightly agitating the tube, without removing it
from the bath.
d. After a further exposure of 10 minutes remove the tube from
the bath and place it immediately in a vessel of ice cold water to
cool. Then incubate at a temperature favorable to the development
of the organism under observation.
e. Raise the temperature of the bath 5 degrees, i. e., to 50° C.,
inoculate another tube. Keep it at 50° for 10 minutes, remove, cool
and incubate.
/. In the same manner expose the organism to the following
temperatures : 55°, 60°, and 65° C. for a period of 10 minutes each.
g. In all cases incubate at least a week and take as the thermal
death point the lowest temperature at which growth fails to appear.
70 GENERAL BACTERIOLOGY
(In more accurate work the temperature should be determined
within 2° C.)
REFERENCES. M. & R. 70 ; McF. 246 ; P. 146 ; P. B. C. 32.
SPECIAL DIRECTIONS. Use B. coli or B. typlwsus.
EXERCISE 42. COMPARATIVE EFFICIENCY OF DRY AND MOIST
HEAT.
GENERAL DIRECTIONS.
a. Charge a water blank with culture of a spore-bearing bacillus,
shaking it well to break up the clumps.
&. Sterilize eight cover-glasses by passing them several times
through the flame, and place four in each of two sterile Petri dishes.
c. With a sterile loop place an equal quantity of the bacterial
suspension (a.) on each cover-glass, and dry by placing Petri dishes
in the incubator with the covers slightly raised.
d. When dry place one Petri dish in the dry sterilizer (near the
thermometer), and the other in the steamer.
e. Keep both sterilizers at a temperature of 100° C., and at the
end of 5, 10, 20 and 40 minutes respectively, remove one cover-glass
from each Petri, place it in a sterile Petri dish and pour a tube of
liquefied gelatin or agar over it. Tip the dish from side to side to
dislodge as many of the bacteria as possible from the cover-glass,
solidify the medium and incubate.
SPECIAL DIRECTIONS. Use an old (spore-bearing) culture of B.
subtilis. Arrange data in the form of a table.
EXERCISE 43. EFFECT OF DESICCATION.
GENERAL DIRECTIONS.
a. Prepare five cover-glasses each of a spore-bearing and a non-
spore-bearing culture, as directed in 42.
&. Place them in sterile Petri dishes, and dry in the incubator.
c. Next morning and every twenty-four hours later plate one of
the cover glasses.
d. In this way determine the length of time the organism in
question can withstand desiccation.
REFERENCES. F. 77 ; L. & X. 40.
SPECIAL DIRECTIONS. Use a young culture of B. coli and an old
(spore-bearing) culture of B. subtilis. Tabulate results.
72 GENERAL BACTERIOLOGY
EXERCISE 44. EFFECT OF CHEMICALS ON BACTERIA.
GENERAL DIRECTIONS.
a. Inoculate three tubes containing 10 cc. of sterile bouillon, with
three loopfuls of a 24-hour old broth culture of organism to be
studied.
&. Add 0.1 cc. of a 5% solution of carbolic acid to one tube (No.
1) ; 0.6 cc. to another (No. 2) ; and 2 cc. to the third (No. 3).
c. Two hours later transfer three loopfuls from each tube to
sterile bouillon and incubate all of the tubes at 38° C.
d. The carbolic acid does not prevent growth in No. 1 or its sub-
culture. In No. 2 no growth, but abundant in its sub-culture (acts
as an antiseptic ) . In both No. 3 and its sub-culture no growth ( acts
as a disinfectant).
REFERENCES. F. 81 ; L. & N. 37 ; L. 107 ; McF. 45.
SPECIAL DIRECTIONS. Use B. coli.
EXERCISE 45. RELATION TO OXYGEN.
GENERAL DIRECTIONS.
a. Pour a tube of melted agar into a sterile Petri dish, and when
the medium has hardened make several parallel streaks with a
platinum loop charged with an aerobic organism.
Z>. Sterilize a piece of mica or a cover-glass, by passing it several
times through the flame and place this over several of the streaks.
This is to shut out the air and should therefore be in perfect contact
with the medium.
c. Make another plate in the same way, using an anaerobe.
REFERENCES. F. 60 ; L. & N. 41 ; L. 180 ; M. & R. 19 ; McF. 212 ;
P. 151.
SPECIAL DIRECTIONS. Use B. subtilis and an anaerobe. Sketch.
EXERCISE 46. EFFECT OF DIRECT SUNLIGHT.
GENERAL DIRECTIONS.
a. Make an agar plate of the organism to be studied (seeding
rather thickly).
&. When agar has thoroughly set, invert the Petri and paste on
under side a piece of black paper from which has been cut out a
number of letters, e. g. student 's initials.
c. Expose this dish, paper side up, to the direct sunlight for a
number of hours (4-6).
GENERAL BACTERIOLOGY
d. Remove the paper and incubate.
REFERENCES. F. 71 ; M. & R. 20 ; L. 77 ; McF. 41 ; P. 135.
SPECIAL DIRECTIONS. Use B. prodigiosus (EHRENB.) FLUEGGE.
Sketch.
EXERCISE 47. DETECTION OF GAS (SHAKE CULTURE).
GENERAL DIRECTIONS.
a. Melt a tube of dextrose agar or dextrose gelatin and inoculate
with a gas-producing organism.
&. Thoroughly mix and solidify by placing in ice water.
c. Incubate over night.
REFERENCES. H. 70 ; L. & N. 89 ; M. & R. 78 ; McF. 49 ; P. 82.
SPECIAL DIRECTIONS. Use B. coli; incubate. Make sketch.
EXEECISE 48. QUANTITATIVE ANALYSIS OF GAS (FERMENTATION
TUBE).
GENERAL DIRECTIONS.
a. Inoculate the open arm of
a fermentation tube with a gas-
producing organism.
b. Incubate at 38° C.
c. By frequent observations
determine :
1. Whether growth takes
place in the open or closed arm,
i. e., whether it is aerobic or an-
aerobic.
2. The rapidity and total
amount of gas formation. Use Frost's gasometer. (Plate I.)
3. Kinds of gas. When the culture has ceased producing gas,
completely fill the open arm with a 2% solution of sodium hydrate;
place the thumb over the mouth of the tube and thoroughly mix the
NaOH with the gas in the closed arm, then without removing the
thumb return the gas to the closed arm, remove the thumb, when the
medium will rise in the closed arm to take the place of the absorbed
C02. Measure. The remaining gas is considered as hydrogen;
bring this into the open arm, remove the thumb and introduce a
lighted match. Air mixed with the hydrogen present causes a
slight explosion. Express the amount of C02 and H. in the form
C02
of a proportion. =^ .
FIG. 19. Fermentation tube, showing
method of using: gasometer.
76 GENERAL BACTERIOLOGY
REFERENCES Smith: Wilder Quarter Century Book, 1893, p.
187 ; A. 212 ; McF. 49 ; M. & R. 79 ; P. 82.
SPECIAL DIRECTIONS. Use B. coli; also try B. subtilis.
EXERCISE 49. DETECTION OF ACIDS AND ALKALIES (WURTZ).
GENERAL DIRECTIONS.
a. Melt a tube of lactose agar (or lactose gelatin) and add enough
of a sterile blue litmus solution (37 e.) to give it a distinct color, cool
to 42° C., inoculate it with an acid-producing organism and pour in
the usual manner.
b. When the agar has solidified invert the dish and place it in
the incubator.
REFERENCE. McF. 51.
SPECIAL DIRECTIONS. Use sewage, putting a drop in a water
blank and using a loop or two of this.
EXERCISE 50. QUANTITATIVE DETERMINATION OF ACIDS.
GENERAL DIRECTIONS.
a. Inoculate 5 test-tubes of dextrose bouillon (or milk) with an
acid-producing organism.
b. Twenty-four hours later remove, with a sterile pipette, 5 cc.
of the medium from one of the tubes and titrate with a twentieth
normal potassium (or sodium) hydrate solution, using phenolphtha-
lien as an indicator.
c. Make titrations as described above on each of the four suc-
ceeding days, using the same amount of culture each day.
d. Plot the results, expressing the number of cc. of hydrate solu-
tion as ordinates and the daily intervals as abscissae.
SPECIAL DIRECTIONS. Use B. coli and incubate at 38° C.
EXERCISE 51. DETECTION OF NITRITES IN CULTURES.
GENERAL DIRECTIONS.
a. Make a culture of a reducing organism in a test-tube of the
nitrate solution (37 g.).
b. Incubate at 28° C. for 1 week, add 1 cc. of each of following
solutions :
1) Sulphanilic acid (para-amido benzenesulphonic acid) 0.5 gm.
Acetic acid (sp. gr. 1.04) 150 cc.
2) a-amido-naphthalene acetate. Boil 0.1 gram of solid a-amido-
naphthalene in 20 cc. of water, filter the solution through a plug of
78 GENERAL BACTERIOLOGY
washed absorbent cotton, and mix the nitrate with 180 cc. of diluted
acetic acid. All water and vessels used must be free from nitrites.
(Leffmann and Beam.)
The presence of a nitrite is indicated by a pink color.
c. A tube of the original medium should be incubated and tested
as a control.
REFERENCES. A. 226 ; McF. 53.
SPECIAL DIRECTIONS. Use sewage,
EXERCISE 52. DETECTION OF AMMONIA.
GENERAL DIRECTIONS.
a. Make bouillon culture and incubate 24 to 48 hours.
&. Place in neck of tube a piece of filter paper which has been
dipped in Nessler's reagent (for formula see works on water analy-
sis). A yellow to reddish brown color indicates the presence of
ammonia.
REFERENCE. L. & N. 78.
SPECIAL DIRECTIONS. Use sewage to inoculate medium.
EXERCISE 53. DETECTION OF SULPHURETTED HYDROGEN.
GENERAL DIRECTIONS.
a. Make a culture in a test-tube, or better, in a flask of bouillon,
and incubate at 38° C.
6. Twenty- four hours later fasten in the flask, by means of the
cotton plug, a strip of filter paper moistened with lead acetate.
c. The presence of sulphuretted hydrogen is indicated by change
of color from brownish to blue. The color change is often slight
and can be best detected by frequent observations.
REFERENCE. L. & N. 76.
SPECIAL DIRECTIONS. Use B. coli or sewage.
EXERCISE 54. DETECTION OF INDOL.
GENERAL DIRECTIONS.
a. Make two cultures in tubes of sugar-free broth (or Dunham's
solution ) .
5. Five days later add a few drops of concentrated sulphuric
acid. The appearance of a pink color indicates that nitroso-indol
has been formed (cholera-red reaction). If the pink or deep red
color does not appear, add 1 cc. of sodium nitrite solution (sodium
80 GENERAL BACTERIOLOGY
or potassium nitrite 0.02 grams and distilled water lOOce.). The
appearance of a red color indicates formation of indol.
REFERENCES. A. 223 ; H. 21 ; L. & N. 142 ; McF. 57 ; M. & R. 80.
SPECIAL DIRECTIONS. Use B. coli or sewage.
EXERCISE 55. DETERMINATION OF CHEMICAL ENZYMES IN CUL-
TURES.
GENERAL DIRECTIONS.
a. Make two gelatin stab cultures of a rapidly liquefying organ-
ism and incubate several days or until the gelatin has all been lique-
fied and then add to each TV cc. of a 5% solution of carbolic acid for
each cc. of medium, shake thoroughly and filter.
5. Pour one into a tube of sterile gelatin and the other into a
tube of milk and note changes.
REFERENCE. McF. 56.
SPECIAL DIRECTIONS. Use B. subtilis or B. prodigiosus.
EXERCISE 56. VARIATION IN ENZYME PRODUCTION.
Make stab cultures of Pseudomonas aeruginosa (SCHROETER)
MIG. (B. pyocyaneus), or any slow liquefier, in ordinary neutral
gelatin and also in dextrose gelatin. Compare rate of liquefaction
in each.
EXERCISE 57. VARIATION IN COLOR PRODUCTION.
Make an agar streak of B. prodigiosus. Incubate at 38° C. ; 24
hours later transfer to fresh media. Continue the process of daily
transplanting from cultures of previous day until chromogenic
property is lost, even at the room temperature.
82 GENERAL BACTERIOLOGY
CHAPTER III
TAXONOMY
In order to become acquainted with a particular organism, to
differentiate it from its congeners or to assign it a definite place in a
system of classification, it must be studied under various conditions
and its characters determined as indicated in the following table.
POINTS TO BE OBSERVED IN THE STUDY OF BACTERIA.
The following scheme gives the most important points to be
noted in the description of an organism, together with some of the
more common descriptive terms suggested by Chester and others.
MORPHOLOGICAL CHARACTERS.
a. Form and arrangement: Spherical, micrococcus, single and
irregularly grouped ; diplococcus, streptococcus, tetracoccus, sarcina,
rods, single, in chains and in filaments ; spirals.
5. Size.
1. In terms of the micromillimeter ; breadth, average and ex-
treme length.
2. In terms of human blood cells.
c. Stain.
1. Aqueous solutions: stains easily or with difficulty; uniformly
or irregularly.
2. Special stains : Gram ; tubercle ; etc.
d. Motility.
1. Brownian movement.
2. Vital movement: sluggish or active; rotary or direct; most
favorable temperature : age ; media : etc.
3. Flagella: stained by Loeffler, Bunge or Van Ermengem's
method ; distribution : monotrichous, lophotrichous or peritrichous.
e. Capsule: stained by Ziehl: Gram or Welch's method; most
favorable conditions ; broad or narrow ; present in serum, milk or on
agar streaks.
GENERAL BACTERIOLOGY
83
/. Spores : time required for formation ; media ; position in cell,
center or end; effect on shape of cell, clostridium, or drumstick;
germination, time, temperature; stain, Hauser or Moeller's method;
temperature limits.
g. Vacuoles (plasmolysis).
h. Crystals.
i. Involution forms.
j. Pleomorphism.
1. Effect of various media.
2. Effect of reaction of media.
CULTURE CHARACTERS.
PLATE- CULTURES (Gelatin and Agar).
I. Surface Colonies.
1. Form: Punctiform, too small to be denned by the naked
eye; circular; oval; fusiform, spindle-shaped, tapering at each end;
cochleate, twisted like a snail shell (Fig. 20, A) ; conglomerate, an
aggregate of similar colonies (Fig. 20, B) ; ameboid, very irregular
like the changing forms of amebae (Fig. 20, C) ; rhizoid, of an
irregular branched root-like character (Fig. 20, D) ; floccose, of a
dense woolly structure; curled, filaments in parallel strands, like
locks or ringlets (Fig. 20, E) ; myceloid, a filamentous colony with
the radiate character of a mould (Fig. 20, F) ; filamentous, an irreg-
ular mass of loosely woven filaments (Fig. 20, G) ; rosulate, shaped
like a rosette.
FIG. 20. Types of Colonies. A. Cochleate (B. coli, abnormal form). B. Conglomerate
(B. Zopfii). C. Ameboid (B. Vulgatus). D. Rhizoid (B. mycoides). E. Curled (B. an-
tnracis). F. Myceloid (B. radiatus). G. Filamentous.
84 GENERAL BACTERIOLOGY
2. Size expressed in millimeters.
3. Surface elevation: Flat, thin spreading over the surface
(Fig. 21, a) ; effused, spreading over the surface as a thin veilly
layer, more delicate than the preceding; raised, thick growth, with
abrupt, terraced edges (Fig. 21,
b) ; convex, surface segment of a
circle, but very flatly convex
(Fig. 21, c) ; pulvinate, surface
segment of a circle, but decided-
ly convex (Fig. 21, d) ; capitate,
hemispherical (Fig. 21, e) ; urn-
J bilicate, shaped like a navel (Fig.
FIG. 21 Surface Elevations of Growths. 21, f ) ; umbonate, bearing a knob
a. Flat: &, Raised; c, Convex; d, Pulvinate; ' '
e, Capitate; f, Umbilicate ; fir, Umbonate. in the Center (Fig. 21, g).
4. Topography of surface: Smooth, surface even without any
of the following distinctive characters ; alveolate, marked by depres-
sions separated by thin walls so as to resemble a honey comb ; punc-
tate, dotted with punctures like pin-pricks ; bullate, like a blistered
surface, rising in convex prominences, rather coarse ; vesicular, more
or less covered with minute vesicles due to gas formation, more
minute than bullate ; verrucose, wart-like, bearing wart-like promi-
nences ; squamose, covered with scales ; echinate, beset with pointed
prominences ; papillate, beset with nipple or mamma-like processes ;
rugose, short, irregular folds due to shrinkage ; contoured, an irreg-
ular but smoothly undulating surface like the surface of a relief
map ; rimose, abounding in chinks, clefts, or cracks.
5. Microscopic structure.
A. Colony as whole: Power of refraction, weak or strong;
amorphous, without definite structure; hyaline, colorless or clear;
homogenous, structure uniform throughout; areolate, divided into
rather irregular or angular spaces by more or less definite boun-
daries (Fig. 22, 1) ; granular, finely or coarsely; grumose, clotted
appearance, particles in clustered grains (Fig. 22, 2) ; moruloid,
having the character of a morula divided into more or less regular
segments (Fig. 22, 3) ; clouded, having a pale ground with ill-defined
patches of deeper tint (Fig. 22, 4) ; gyrose, marked by wavy lines
indefinitely placed (Fig. 22, 5) ; rivulose, marked by lines like the
rivers of a map ; rimose, showing chinks, cracks or clefts ; marmo-
rated, showing faint, irregular stripes, or traversed by vein-like
markings as in marble (Fig. 22, 6) ; reticulated, in the form of a
GENERAL BACTERIOLOGY
85
network, like the vein of a leaf (Fig. 22, 7) ; filamentous, floccose, or
curled, as defined under 1 above.
FIG. 22. Microscopic Structure of Colonies. A, Colony as a whole. B, Edge of Colony.
1, Areolate; 2, Grumose; 3, Moruloid; 4, Clouded; 5, Gyrose; 6, Marmorated; 7, Recticu-
Jate; 8, Repand; 9. Lobate; 10, Erose; 11, Auriculate; 12, Lacerate; 13. Fimbricate; 14,
Ciliate.
B. Edge of colonies: entire, without toothing or division;
undulate, wavy; repand, like the border of an open umbrella (Fig.
22, 8) ; lobate, (Fig. 22, 9) ; erose, as if gnawed, irregularly toothed
(Fig. 22, 10) ; auriculate, with ear-like lobes (Fig. 22, 11) ; lacerate,
irregularly cleft, as if torn (Fig. 22, 12) ; -/imbricate, fringed (Fig.
22, 13) ; ciliate, hair-like extensions, radially placed (Fig. 22, 14) ;
filamentous, (Fig. 20, G) ; curled, (Fig. 20, E).
6. Color (to be determined for both transmitted and reflected
light) : transparent; vitreous, transparent and colorless; oleaginous,
transparent and yellow, olive to linseed oil colored ; resinous, trans-
parent and brown, varnish or resin colored : translucent; paraffinous,
translucent and white, porcelanous ; opalescent, translucent, grayish-
white by reflected light, smoky-brown by transmitted light ; nacreous,
translucent, grayish- white with pearly lustre; sebaceous, translu-
cent, yellowish or grayish white, tallowy ; butyrous, translucent and
yellow; ceraceous, translucent and wax colored; opaque; cretaceous,
opaque and white; chalky, dull without lustre; glossy, shining;
fluorescent; iridescent.
7. Consistency: hard, friable; soft; viscid.
8. Changes in medium: Liquefaction (gelatin), shape of liqui-
fied area, character of the fluid, membrane and sediment see under
Bouillon below ; color; odor; consistency.
II. Deep Colonies.
1. Form. 2. Size. 3. Character of surface. 4. Microscopic
structure. 5. Consistency. 6. Changes in medium. Same as sur-
face colonies.
STAB CULTURES (Gelatin or Agar).
I. Non-liquefying.
86
GENERAL BACTERIOLOGY
1. Line of puncture : filiform, uniform growth without any spe-
cial characters (Fig. 23, 1) ; nodose, consisting of closely aggregated
colonies; beaded, loosely placed or disjointed colonies (Fig. 23, 2) ;
papillate, covered with papillae; echinulate, minutely prickly (Fig.
23, 3) ; villous, beset with undivided hair-like extensions (Fig. 23,
4) ; plumose, a delicate feathery growth; arborescent, beset with
branched hair-like extensions (Fig. 23, 5).
2. Surface growth. Same as for plate cultures.
B
FIG. 23. Types of Growth in Stab Cultures. A, Non-liquefying: 1, Filiform (B. coli);
2. Beaded (Str. pyogenes); 3, Echinate (Bact. acidi-lactici) ; 4, Villous (Bact. murisepti-
cum); 5, Arborescent (B. mycoides). B, Liquefying: 6, Crateriform (B. vulgais, 24
hours); 7, Napiform (B. subtilis, 48 hours); 8, Infundibuliform (B. prodigiosus); 9, Sac-
cate (Msp. Finkleri): 10, Stratiform (Ps. fluorescens).
II. Liquefying.
1. Shape of liquefied area: crateriform, saucer shaped (Fig.
23, 6) ; napiform, outline of a turnip (Fig. 23,7) ; infundibuliform,
shape of a funnel, conical (Fig. 23, 8) ; saccate, shape of an elon-
gated sac (Fig. 23, 9) ; stratiform, liquefaction extending to the
walls of the tube and then downward horizontally (Fig. 23, 10).
2. Condition of fluid : See Bouillon below.
STREAK CULTURES (Gelatin, Agar, Potato or Blood serum).
1. Form: filiform (Fig. 24, 1) ; nodose; beaded (Fig. 24, 3) ;
GENERAL BACTERIOLOGY
papillate; echinulate (Fig. 24, 2) ; effused (Fig. 24, 4) ; villous;
plumose; arborescent (Fig. 24, 5).
FIG. 24. Types of Streak Cultures: 1, Filiform (B. coli); 2, Echinulate (Bact. acidi-
lactici); 3, Beaded (Str. pyogenes); 4, Effuse (B. vulgaris) ; 5, Arborescent (B.mycoides).
2. Size ; in millimeters.
3. Surface elevation.
4. Topography of surface.
5. Color.
6. Consistency.
7. Changes in medium.
>- Same as plate cultures.
BOUILLON CULTURES.
1. Condition of fluid : clear; clouded, degree of, does or does not
clear on standing.
2. Membrane: when formed; color; consistency; structure.
3. Sediment : amount; color; character; whether compact or
flocculent; on agitation appears granular, flaky or viscid.
4. Reaction.
MILK CULTURES.
I. Curd formed:
1. Time required to curdle.
2. Character of curd: hard or soft1; massed or in fragments;
changed or not on boiling.
3. Whey : amount; transparent or turbid.
4. Reaction : effect on litmus.
5. Digestion: time required; solution complete or incomplete;
reaction; character of solution, clear, or cloudy.
6. Gas bubbles.
7. Odor.
II. Digestion without formation of curd.
III. No visible change even after boiling.
88 GENERAL BACTERIOLOGY
PHYSIOLOGICAL CHARACTERS.
a. Effect of desiccation.
fe. Relation to temperature: minimum; optimum; maximum;
thermal death point.
c. Relation to oxygen : under mica plate ; in hydrogen or nitrogen.
d. Relation to light, Buchner's Experiment (46).
e. Relation to antiseptics and disinfectants.
/. Pigment production : relation of development to oxygen ; rela-
tion of development to character of medium ; changes produced by
alkali and acid ; solubility ; spectrum analysis.
g. Gas production : rate, quantity and formula produced in dex-
trose, lactose, and saccharose media.
h. Acid and alkali production: carbohydrates present; carbo-
hydrates absent.
i. Relation of growth to acidity and alkalinity of medium ; growth
in 1, 2, 3 and 4% alkali ; growth in 1, 2, 3, 4 and 5% acid.
j. Reduction of nitrates : to nitrites ; to ammonia. '
Jc. Production of sulphuretted hydrogen.
Z. Production of indol in sugar-free bouillon.
m. Enzyme production ; proteolytic ; diastatic.
n. Characteristic odor.
o. Pathogenesis :
1. Modes of inoculation by which its pathogenic properties are
demonstrated.
2. Quantity of material required.
3. Duration of the disease and its symptoms.
4. Lesions produced and the distribution of the bacteria in
the inoculated animals.
5. Which animals are susceptible and which are immune.
6. Variations in virulence and the probable causes to which
they are due.
7. Detection of toxic or immunizing products of growth.
8. Agglutinating properties of serum of immune animals.
(Widal reaction.)
9. Lysogenic properties of serum of immune animals.
( Pf eiff er 's phenomenon. )
REFERENCES. Chester, Reports Delaware Experiment Station,
1897, 1898 and 1899; A. 227; C. 17; H. 105; P. B. C. (Cheesman's
Charts) ; Kendall, Rept. Am. Pub. H. Assn., 28: 481.
GENERAL BACTERIOLOGY
89
MIGULA'S SYSTEM OF CLASSIFICATION.
I. Cells globose in a free state, not
elongated in any direction be-
fore division into 1, 2, or 3
planes. COCCACEAE ZOPF emend. MIG.
A. Cells without organs of mo-
tion.
a. Division in one plane,
6. Division in two planes,
c. Division in three planes,
1. Streptococcus BILLROTH.
2. Micrococcus (HALLIER )
COHN.
3. Sarcina GOODSIR.
4. Planococcus MIGULA.
5. Planosarcina MIGULA.
II. Cells cylindrical, longer or
shorter, and only divided in
one plane, and elongated to
twice the normal length be-
fore the division.
(1) Cells straight, rod-shaped
without sheath, non-
motile or motile by
means of flagella. BACTERIACEAE MIGULA.
A. Cells without organs of
motion,
B. Cells with organs of mo-
tion (flagella).
a. Flagella distributed over
the whole body,
6. Flagella polar,
|(2) Cells crooked, without
sheath. SPIRILLACEAE MIGULA.
X Cells rigid, not snake-like
or flexuous.
a. Cells without organs of
motion (flagella), - 9. Spirosoma MIGULA.
b. Cells with organs of
motion (flagella).
1. Cell with 1, very rare-
ly 2-3 polar flagella, 10. Microspira SCHROETER.
- 6. Bacterium EHRENB.
7. Bacillus COHN.
8. Pseudomonas MIGULA.
90
GENERAL BACTERIOLOGY
2. Cells with polar fla-
gella- tufts, - -11. Spirillum EHRENB.
B. Cells flexuous, - - 12. Spirochaeta EHRENB.
(3) Cells inclosed in a sheath. CHLAMYDOBACTERIACEAE
MlGULA.
A. Cell contents without gran-
ules of sulphur.
a. Cell threads unbranched.
1). Cell division always
only in one plane, - 13. Streptothrix COHN.
2 ) . Cell division in three
planes previous to
the formation of co-
nidia.
i). Cells surrounded
by very delicate,
scarcely visible
sheath (marine).
ii). Sheath clearly
visible ( fresh wa-
ter), -
&. Cell threads branched,
B. Cell contents containing sul-
phur granules.
14. Phragmidiothrix ENG-
LER.
15. Crenothrix COHN.
16. Cladothrix COHN.
17. Thiothrix WINOGRAD-
SKY.
(4) Cells destitute of a sheath,
united into threads mo-
tile by means of an un-
dulating membrane. BEGGIATOACEAE TREVISAN.
Only one genus, - - - 18. Beggiatoa TREVISAN.
GENERAL BACTERIOLOGY 91
BACTERIA ARRANGED IN CLASSES AND GROUPS.
Saprophilic Class:
Bacillus vulgatus Trevisan.
Bacillus subtilis (Ehrenb.) Cohn.
Chromogenic Class :
Bacillus prodigiosus (Ehrenb.) Fluegge.
Zymogenic Class :
Bacterium acidi-lactici Hueppe.
Saprogenic Class :
Bacillus vulgar is (Hauser) Mig. .
Bacillus Zopfii (Kurth) Mig.
Phosphorescent Class :
Bacterium phosphorescens (Cohn) Fischer.
Pathogenic Aerobes.
Erysipelas Group :
Streptococcus erysipelatos Fehleisen.
Pus Coccus Group :
Micrococcus pyogenes var. albus (Rosenbach) L. & N.
Micrococcus pyogenes var. aureus (Rosenbach) L. & No
Malta Fever Group :
Micrococcus melitensis Bruce.
Diplococcus Group :
Micrococcus gonorrhoeas (Baum) Fluegge.
Micrococcus Weichselbaumii (Trevisan).
Sarcina Group :
Sarcina tetragena (Gaffky) Mig.
Anthrax Group :
Bacterium anthracis (Koch) Mig.
Friedlander Group:
Bacterium pneumonicum (Fried.) Mig.
Bacterium aerogenes (Esch.) Mig.
Bacterium capsulatum (Sternberg) Chester.
Swine Plague Group :
Bacterium choleras (Zopf ) Kitt.
Bacterium bovisepticum (Kruse) Mig.
Glanders Group:
Bacterium mallei (Loeffler) Mig.
Bacterium rhusiopathiag (Kitt) Mig.
92 GENERAL BACTERIOLOGY
Diphtheria Group :
Bacterium diphtheriae (Loeffler) Mig.
Bacterium pseudodiphtheriticum (Loeffler) Mig.
Pneumonia Group :
Bacterium pneumonias (Weichsel.) Mig.
Influenza Group :
Bacterium influenzas (Pfeiffer) Lehm. and Neum.
Tubercle Group :
Bacterium tuberculosis (Koch) Mig.
Bacterium tuberculosis var. avium (Kruse) Mig.
Colon Group :
Bacillus coli (Escherich) Mig.
Bacillus enteritidis Gaertner.
Hog Cholera Group :
Bacillus Salmonii (Trevisan) Chester.
Bacillus icteroides Sanarelli.
Typhoid Group :
Bacillus typhosus Zopf .
Bacillus dysenteriae Shiga.
Bacillus pestis Lehmann and Neumann.
Pseudomonas Group :
Pseudomonas aeruginosa (Schroeter) Mig.
Cholera Group :
Microspira comma (Koch) Schroeter.
Microspira Metschnikovi (Gamaleia) Mig.
Microspira Schuylkilliensis (Abbott) Chester.
Streptothrix Group :
Streptothrix bovis (Harz) Chester.
Streptothrix Madurae Vincent.
Pathogenic Anaerobes.
Emphysema Group:
Bacterium Welchii Mig.
(Edema Group :
Bacillus Feseri (Trevisan) Chester.
Bacillus edematis Zopf.
Bacillus botulinus v. Ermengem.
Tetanus Group :
Bacillus tetani Nicolaier.
GENERAL BACTERIOLOGY 93
CHAPTER IV
SYSTEMATIC STUDY OF REPRESENTATIVE
NON-PATHOGENIC BACTERIA
In making a systematic study of a bacterium it is necessary to
determine as many as possible of the points indicated in the previous
chapter (III.) ; and in the laboratory this becomes a regular rou-
tine procedure— in the study of each germ. The organism is first
inoculated into a number of the standard media. These cultures are
frequently spoken of as a "set of cultures" and are usually com-
posed of the following : Gelatin and agar plates, a gelatin stab, agar
and potato streaks, a bouillon culture (or Dunham's sol.), a milk
culture and a dextrose gelatin or agar stab (or shake culture).
These cultures are then incubated at the proper temperature for 24
hours. They are then examined, described and sketched. At the
same time three cover-glass preparations are made, one each from
the agar, bouillon and gelatin cultures and stained with the follow-
ing dyes: agar with an aqueous solution, bouillon with Loeffler's
methylen blue, and the gelatin by Gram's method. The bouillon
culture is also examined in a hanging-drop for motility and the milk
culture for capsules. From these microscopical preparations the
morphological characters can usually be determined. The cultures
are again placed in the incubator and 24 hours later (48 hours after
inoculation) are again examined and any changes are noted and
sketched. The cultures are now usually kept at the temperature
of the room for about one week and then examined for the last
time.
If the organism produces gas in dextrose media, fermentation
tubes should be inoculated and the rate, amount and formula of the
gas determined.
The descriptions and sketches are conveniently made on the
charts provided on the following pages.
94 GENERAL BACTERIOLOGY
EXERCISE 58. PREPARATION OF SPECIAL MEDIA.
Tube and sterilize the following media for work in Chapters IV,
and V. :
80 tubes of ordinary or nutrient agar.
2 tubes of lactose agar.
10 tubes of dextrose agar or gelatin.
20 tubes of gelatin.
10 tubes of bouillon.
10 fermentation tubes of dextrose bouillon.
10 tubes of potato.
10 tubes of milk.
10 tubes of sugar-free bouillon, or Dunham 's solution,
10 water-blanks.
EXERCISE 59. SAPEOPHILIC CLASS. 95
Bacillus vulgatus Trevisan.
SYNONYMS. Bacillus mesentericus vulgatus Fluegge ; Potato
bacillus.
EXPLANATORY. This is a widely distributed organism which was
first described by Fluegge in 1886. Its spores are very resistant and
can almost invariably be found on potatoes. It can usually be ob-
tained by boiling potatoes for a half an hour, halving them and incu-
bating in a sterile moist chamber.
REFERENCES. Fluegge : Die Mikroorganismen, 1886 ; C. 271 ; L.
& N. 323 ; Mig. 2 : 556.
MORPHOLOGICAL CHARACTERS :
1. FORM AND ARRANGEMENT:
a. Bouillon
SKETCHES.
&. Agar
c. Gelatin.
d. Other media.
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet. .
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
&. Flagella stain
5. SPORES:.
6. SPECIAL, CHARACTERS: —
a. Capsules
&. Involution forms
c. Deposits or vacuoles.
d. Pleomorphism
96 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -j- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48hoursat... ...°C. 6 days at °C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. : (b) Deep Colonies.
Sketches.
°c
«c.
Special Media: (Such as litmus milk and blood serum j
BACILLUS VULGATUS
97
Gelatin Stab: Grown24hours at °C
?
48 hours at ,
6 days at oC,
Agar Streak: Grown 24 hours at °C.
48 hours at °C. 6 days at °C.
Potato: Grown 24 hours at , °C.
b3
I
48 hours at °C. 6 days at °C.
! '
Bouillon: Grown 24 hours at °C. Q
o
t§
I \ )
48 hours at ,°C. 6 days at °C.
98 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture :
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COz: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:...
litmus milk
7. REDUCTION OF NITRATES:.
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
SAPROPHILIC CLASS— CONTINUED.
99
Bacillus subtilis (Ehrenb.) Cohn.
SYNONYMS. Vibrio subtilis Ehrenberg ; Hay bacillus.
EXPLANATORY. This is a well-known and widely distributed or-
ganism. First described by Cohn in 1872. It is almost invariably
found on hay, hence the common name. Its spores, like those of the
''potato bacillus," are very resistant to heat. A pure culture can
usually be obtained by making an infusion of hay or straw and
heating it to 80° C. for ten minutes.
REFERENCES. F. Cohn, Beitraege Zur Biologic, Bd. I, 1872,
Heft 2, p. 175 ; C. 276 ; L. 170 ; L. & N. 317 ; Mig. 2 : 515.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
&. Flagrella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
100 CULTURE CHARACTERS
Reaction of media (Fuller's scale) -j- or —
Gelatin plate: Grown 24 hours at 'C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6days at °C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media1. (Such as litmus milk and blood serum.)
BACILLUS; SUBTILIS \ ' V ' i '*'. : 5 : f 1 0 L
Gelatin Stab: Grown 2A hours at °C. Q Q
o o
ti
cc
h
48 hours at °C. 6 days at 0C.
Agar Streak: Grown 24 hours at *C= Q Q
' /\
/ \
c, ,
2 £
§ I §
,8
/I K
\
48 hours at °C. 6 days at °C.
Potato: Grown 24 hours at °C. jj | Q
/\
I
A^,^
48 hours at °C. 6 days at °C
Bouillon: Grown 24 hours at °C. ^ Q
48 hours at °C.
6 days at °C.
? » * »«
rs -O -i-T'i •-•-,*,•»•" - n-r i- » "i1- o v
-1 ".-"> ?" -j v- -l " i'1 » » *="* "> -"• »
102 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: ,.
optimum °C.; limits to °C.;
thermal death-point °C.: time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3 RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H : CO2: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION: . . . . ,
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia...
8. INDOL PRODUCTION
48 hours days
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) ;
EXERCISE 60. C1IROMOGKENIC CLASS. 1Q3
Bacillus prodigiosus (Ehrenb.) Fluegge.
SYNONYMS. Monas prodigiosa Ehrenb.; M. prodigiosus Cohn.
EXPLANATORY. This organism was first described by Ehrenberg. It is
the oldest known chromogenic bacterium. It is very commonly found in the
air of Europe and has a very interesting history on account of its casual
relation to bread epidemics — ''bloody bread," "bleeding host," etc. It
occurs spontaneously in this country. It is slightly pathogenic. Introduced
intraperitoneally into guinea pigs in large quantities it produces death. In-
oculated into animals naturally immune to malignant oedema it renders them
susceptible. Rabbits inoculated with anthrax are protected by a subsequent
inoculation with this organism. It is grown with the streptococcus of erysip-
elas to produce Coley's Fluid for treatment of inoperable malignant tumors.
REFERENCES. Ehrenberg, Verhandlunger der Berliner Akademie, 1839; C.
258; L. 137; L. & N. 272; Mig. 2: 845.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar.
c. Gelatin.
d. Other media. ,
2. SIZE:
3'. STAINING POWERS :
a Aqueous gentian- violet..
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement. .
6. Flagella stain
5. SPORES:.
SPECIAL CHARACTERS :
a. Capsules
6. Involution forms
c. Deposits or vacuoles..
*?. Pleomorphism . .
104 CULTURE CHARACTERS
Eeaction of media (Fuller 's scale) -j- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches
48 hours at °C.
6 days at °C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at
...on
«c.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS PRODIGIOSUS
105
Gelatin Stab: Grown 24 hours at °C.
48 hours at °C.
6 days at 0C.
Agar Streak: Grown 24 hours at °C.
D
/
48 hours at
6 days at °C.
Potato : Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
Bouillon: Grown 24 hours at °0.
48 hours at *C.
Gdaysat °
106 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: ,.
optimum °C. ; limits to °C. :
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3- RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development : 24 hours per cent. , 48 hours per cent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CCb: : : ,
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk .
7. REDUCTION OF NITRATES:
to nitrites to ammonia...
8. INDOL PRODUCTION
48 hours days .
9. ENEYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) : .
GENERAL BACTERIOLOGY
107
EXERCISE 61. VARIETY OF PIGMENTS.
Make agar or potato streak cultures of the following organisms, incubate
at 28° C., study, describe and sketch.
3 TO 6 DAYS
SKETCHES
Bacillus
indicus
or
Sarcina
aurantiaca
or
Sarcina
lutea
or
Pseudomonas
fluorescens
(B. fluorescens)
Pseudomonas
aeruginosa
(B. pyocyaneus)
Pseudomonas
violacea
or
108 GENERAL BACTERIOLOGY
EXERCISE 62. SEPARATION OF BACTERIAL COLORING MATTER.
a. Make four agar streaks of Bacillus prodigiosus, which are to
be kept in the dark until the coloring matter is well formed.
&. Add about 10 cc. of ether to each tube and shake vigorously
until the red pigment has all been dissolved out.
c. Pour into a large test-tube and allow to stand over night in
the dark, then pipette off the colored portion.
d. Divide this into four parts and treat them as follows :
1. Evaporate on glass slide and examine crystals formed
under microscope.
2. Add a few drops of hydrochloric acid, drop by drop
3. Add a few drops of sodium hydroxide.
4. Stand in direct sunlight.
EXERCISE 63. ZYMOGENIC CLASS. 109
Bacterium acidi-lactici Zopf.
COMMON NAME. Lactic acid bacillus.
EXPLANATORY. This organism may be taken as a type of the
bacteria causing sour milk, of which there are a very large number.
It was first described by Hueppe in 1884. It is very widely dis-
tributed.
REFERENCES. Hueppe, Mitteil. aus dem Kaiserl. Gesundheits-
amte, 1884, Bd. II. p. 1837 ; C. 149 ; Cn. 189 ; L. 222 ; L. & N. 220.
Mig. 2 : 327.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar.
c. Gelatin. .
d. Other media.
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet..
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
i. MOTILITY :
a. Character of movement..
b. Flagella stain
5. SPORES-
G. SPECIAL CHARACTERS: —
a. Capsules
6. Involution forms
c. Deposits or vacuoles.
d. Pleomorphism .
110 CULTURE CHARACTERS
Reaction of media (Fuller 's scale) _|_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surf ace Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C. 6 days at
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM ACIDI-LACTICI
111
Gelatin Stab: Grown 24 hours at °C.
48 hours at
6 days at oC.
Acar Streak: Grown 24 hours at °€.
48 hours at °C.
6 days at °C.
Potato : Grown 24 hours at °C.
48 hours at
6 days at °C.
Bouillon: Grown 24 hours at.... °C.
48 hours at .
• °C. 6 day sat..
112 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATUKE:
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H:CO2:: :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) : . . .
EXERCISE 64. SAPROGENIC CLASS.
Bacillus vulgaris (Hauser) Mig.
113
SYNONYMS. Proteus vulgaris Hauser. B. proteus Trevisan.
EXPLANATORY. First described by Hauser. It is widely dis-
tributed and is commonly found in putrefactive substances. It is
one of several related species included under the old name of ' ' Bac-
terium termo." While in small doses and under ordinary condi-
tions it is harmless, at times, and in large doses, it may be patho-
genic.
REFERENCES. Hauser, Ueber Faulnisbakterien, 1885 ; C. 244 ;
Lafar, 194-199. Mig. 2 : 707.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet. .
&. Loeffler's methylen-blue .
c. Gram's stain
d. Special stains
4. MOTILITY:
a. Character of movement. .
6. Flagella stain
5. SPORES :
€. SPECIAL CHARACTERS:
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
114 CULTURE CHARACTERS
Reaction of media (Fuller's scale) -|- or —
Gelatin plate: Grown 24 hours at °C.
(») Surface Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at *C.
(a) Surf ace Colonies.
Agar plate: Grown 24 hours at °C.
! (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at 'C.
Special Media.: (Such as litmus milk and blood serum.)
BACILLUS VULGARIS
115
Gelatin Stab: Grown 24 hours at °C. Q Q
O O
3 15
£ £
I I
v .)
48 hours at °C. 6 days at
A gar Streak: Grown 24 hours at °C. Q
A ,
£ £ (
§1
A I
\
48 hours at °C. 6 days at °C.
Potato: Grown 24 hours at ... o °C. Q
O
1
ii j i
48 hours at °C. 6 days at. °C.
Bouillon: Grown 24 hours at °C. Q Q
48 hours at °C.
6 days at °C.
116 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C.; limits to °C.;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN :
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. s PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm — closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent .- hours per cent.
reaction in open arm
gas formula, H: COz: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk .
7. REDUCTION OP NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION .
48 hours days
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic...
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) ;
SAPROGENIC CLASS— CONTINUED.
Bacillus Zopfii (Kurth) Mig.
117
SYNONYMS. Bacterium Zopfii Kurth; Proteus Zenkeri Hauser|
EXPLANATORY. This organism belongs to the group of putre-
factive bacteria ( proteus group ) .
REFERENCES. Kurth, Botan. Zeitung, 1883 ; C. 248 ; Mig. 2 : 815.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. A gar
c. Gelatin
d. Other-media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
6. Loeffler's methylen-blue.
c. Grain's stain
d. Special stains
4. MOTILITY :
a. Character of movement. .
b. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
b. Involution forms
c. Deposits or vacuoles ,
d. Pleomorphism
118 CULTURE CHARACTERS
Reaction of media (Fuller 's scale) _ ...
or
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at »C.
(a) Surf ace Colonies.
Agar plate: Grown 24 hours at °C.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at *C.
Special Media: (Such as litmus milk ami blood serum.)
BACILLUS ZOPFII
119
Gelatin Stab: Grown 24 hours at °C.
48 hours at °C.
6 days at oC.
Agar Streak: Grown 24 hours at °C.
48 hours at °C
Potato: Grown 24 hours at °C.
48 hours at
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
120 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours per cent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H: CCb: : :
1>. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic. . .
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 65. PHOSPHORESCENT CLASS. 121
Bacterium phosphorescens (Cohn) Fischer.
SYNONYMS. Photobacterium phosphorescens Beijerinck.
EXPLANATORY. First described by Fischer in 1887. Found in Kiel harbor
on dead sea fish, oysters and occasionally on meat in shops. The production
of light is shown in the dark, especially when the organism is grown on a
medium made by boiling two salt herrings in a liter of water, adding 100
gms. of gelatin to the filtrate without neutralization, tubing and then ster-
ilizing (Lehmann). Phosphorescence can even be restored to attenuated
cultures by growth on this medium. Inasmuch as oxygen is necessary to
light production surface growths are best.
EEFERENCES. Fisher, Zeitschrift fur Hygiene, 1887, Band 2, p. 92; C. 181;
L. &N. 231; Mig. 2: 433.
MORPHOLOGICAL CHARAQTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agrar.
c. Gelatin.
d. Other media..
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
b. Loemer's methylen-blue .
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement. .
6. Flagrella stain
5. SPORES:.
6. SPECIAL CHARACTERS:....
a. Capsules
6. Involution forms
c. Deposits or vacuoles..
d. Pleomorphism,
122 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -|- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at..,.,.... °C.
6 days at., «C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
*8 hours at °C.
6 days at 0C.
Special Media: (Suck as litmus milk and blood serum,;
BACTERIUM PHOSPHORESCENS
123
Gelatin Stab: Grown 24 hours at.... ...,°C.
48 hours at
'C.
6 days at »C.
Agar Streak: Grown 24 hours at °C.
I
48 hours at
6 days at. .
Potato: Grown 24 hours at °C.
48 hours at ° C.
6 days at °C.
Bouillon: Grown 24 hours at °C r-\
4T
i
o
Q
CO
1
48 hours at °C. 6 days at. . .
C.
124 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C.;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN : . . . .
3. RELATION TO OTHER AGENTS, SUCH AS....
desiccation, light, disinfectants, etc.
4. PIGMENT PRODUCTION:
5 GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COz: : :
1). lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES: ..
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin . ... oCigestion of casein.
diastatic ,
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
126 GENERAL BACTERIOLOGY
CHAPTER V
BACTERIOLOGICAL ANALYSIS
EXERCISE 66. COMPARATIVE ANALYSIS OF AIR (Kocn).
a. Plate three tubes of gelatin and expose by removing lid for
20 minutes in the following places: 1. Laboratory. 2. Cellar.
3. Out of doors.
~b. Replace the lids and keep plates at 22° C. for several days.
c. Count the colonies. The counting is facilitated by the use of
Plate II. on which the Petri dishes are to be placed. In counting
a hand lens magnifying about 5 diameters should be used. Where
possible all of the colonies on the plate should be counted, if this
be impossible count a representative area and estimate the whole
number.
d. Express the results in terms of the number of organisms
which fall per square foot per minute. The area of the Petri dish
can be read off directly from Plate II. in square centimeters, or it
can be calculated by multiplying the square of the diameter by
0.785.
This method enables one to make a rough comparison of the
number of organisms occurring in the localities examined, but to
determine the number per volume the following method must be
employed.
REFERENCE. H. 477.
EXERCISE 67. QUANTITATIVE DETERMINATION OF NUMBER OF
BACTERIA IN AIR (PETRI-SEDGWICK).
GENERAL DIRECTIONS.
a. A piece of glass tubing 6 mm. (14 in.) in diameter by 15 cm.
(6 in.) long is drawn out at one end in a gas flame and sealed.
ft. Fill this tube about one-third full with granulated sugar,
insert a cotton plug next to the sugar, and one at the end of the tube
(Fig. 25, A). '
128
GENERAL BACTERIOLOGY
hours at 130'
c. Sterilize in the hot air sterilizer for 1 and
C. (sugar melts at a higher
temperature).
d. Fasten the tube,
pointed end up, in a clamp,
remove the first cotton
plug and connect with an
aspirator (Fig. 26).
e. Break off the pointed
end of the tube and draw a
measured quantity of air
through the sugar.
SPECIAL DIRECTIONS.
a. Filter 50 liters of
air- FIG. 26. Aspirator for
ft. Dissolve SUgar in 10 filtering air.
cc. of sterile water (water-blank) and make
FIG. 25. Apparatus f or fli- plates, using 1 cc. of the mixture.
tering air through sugar. r ,
A, ready for sterilization; c. Incubate, count colonies as above and es-
B, point broken off and at- . .
tached to aspirator; a and timate the number of organisms per liter or air.
6, cotton plugs; c, sugar;
d, clamp; e, rubber tube. , __
KEFERENCES. A. 604; H. 477; L. & K.
392 ; M. & R. 123 ; McF. 230.
EXERCISE 68. WATER ANALYSIS.
COLLECTION. Water for analysis must be
collected in a sterile vessel. A test-tube or flask
may be used in the laboratory, but when the
collection is made outside a sterile glass-stop-
pered bottle should be used. In collecting,
special care should be taken to get a fair sample ;
if the water be in a reservoir, or the like, the
bottle should be filled below the surface to avoid
the scum and away from the bottom to avoid the
sediment. Fig. 27 shows a form of apparatus
used to take samples in deep water. If some
time must necessarily elapse between the collec-
tion of the sample and its examination it should
be packed in ice. Specially constructed shipping
cases are used in most laboratories.
o
FIG. 27. Russell's
Water Sampler, a,
test-tube from which
the air has been ex-
hausted; &, glass tube,
sealed; c, sinker; d,
weight to be dropped
at proper depth.
130 GENERAL BACTERIOLOGY
QUANTITATIVE ANALYSIS.
a. After shaking the sample at least 25 times remove 1 cc. of
the water by means of a sterile pipette and place it in the bottom of a
sterile Petri dish. In the same way remove % cc. and TV cc. Pipettes
graduated to TVths. may be used, or a 1 cc., or even a 5 cc. pipette
may be used by counting the whole number of drops delivered and
then taking the number of drops to make the required fraction. If
the sample be supposed to be highly infected it should be diluted
with sterile water before the cultures are made. Plates ought not
to contain over 200 colonies.
&. Pour into the dishes fluid gelatin (not warmer than 42° C.)
and tip them from side to side until the medium and water are
thoroughly mixed. Solidify and incubate at 22° C., or below.
c. In the same way make agar plates using ordinary agar or,
better, 5% glycerine asrar. Incubate at 22° C.
d. Count the colonies at the end of 48 hours as directed above
(66c.) and at intervals afterward until the maximum number of
colonies is obtained. Express the results in the number of bacteria
per cc. of watei*.
QUALITATIVE ANALYSIS.
a. Number of species. Examine carefully, under the low power
of the microscope, the plates made above to determine the number
of different species, describing each very briefly. Estimate also the
total number of liquefying organisms per cc.
&. Tests for Fecal Bacteria (B. coli) as follows:
1. Fermentation tube test. Inoculate three fermentation tubes,
containing 1% dextrose bouillon, with -j^, 1, and 10 cc. of water
and incubate at 38° C. Tubes which develop from 30 to 70% of
gas should have lactose litmus agar plate cultures made from them
and then the gas formula may be determined. For B. coli it will
be about : C02 : H : : 1 : 2 or CO, _ ^
H =Y
2. Indol test. Tubes of sugar-free bouillon or Dunham's solu-
tion inoculated and incubated at 38° C. for 4 to 5 days will show
the presence of indol if B. coli be present.
3. Acid colonies. A lactose litmus agar plate should be made
132
GENERAL BACTERIOLOGY
(using about 1 cc. of water) also one from fermentation tube and
kept at 38° C. Examine 24 hours later for acid colonies.
c . Pathogenic Bacteria. See Chapter X.
REFERENCES. A. 579 ; H. 457 ; McF. 234 ; M. & R. 133 ; P. 245 ;
Prescott & Winslow, Elements of Water Bacteriology. For the
determination of the various species present see Frankland's Micro-
organisms of Water ; Fuller : Keport Am. Public Health Assoc., 1899,
580; Chester.
SPECIAL DIRECTIONS. Analyze a surface water (lake or river)
and a deep well or a spring water.
EXERCISE 69. ESTIMATION OF NUMBER OF BACTERIA IN SOIL.
a. With a sterile knife collect a sample of soil in. a
sterile test-tube or Petri dish. Samples at various
depths can be secured by means of an earth borer.
(Fig. 28.)
&. Weigh out 1 gram and dilute 1000 times with
sterile water.
c. Make three gelatin plate cultures using 1 cc.,
% cc. and TV cc. of this suspension. Incubate.
d. Count the colonies as they develop and estimate
the number of bacteria per gram of soil.
e. Many of the bacteria of the soil are anaerobic
and can only be grown in the absence of free oxygen.
See Part II. Chap VII. for methods of cultivation.
REFERENCES. A. 609 ; H. 481 ; M. & R. 128 ; McF. FIG. 28.
240 ; Cn. 3. e1'8 Soii
EXERCISE 70. QUANTITATIVE ANALYSIS OF MILK.
a. Obtain a sample of milk in a sterile vessel.
6. Dilute milk 1000 times with sterile water.
c. Make plates as under soil (69).
d. Count colonies and estimate number of bacteria per cc.
EXERCISE 71. EFFICIENCY OF PASTEURIZATION.
a. Place same milk as used in previous experiment in the bot-
tles of a pasteurizing apparatus, such as Freeman's, and pasteurize
as per printed directions. Or, place the milk in ordinary milk bot-
134
GENERAL BACTERIOLOGY
>V-/=J
ties or fruit jars, filling to a uniform level; these are then to be
placed in a flat bottomed pail
(Fig. 29) which is to be filled
with water and heated to 71°
C. (160° F.). Remove source
of heat, cover and allow tc
stand 30 minutes. Remove
bottles and cool as quickly as
possible without danger to
glass. FIG. 29. Home-Made Pasteurizers.
~b. Determine bacterial content of pasteurized product by mak-
ing plates. A dilution of 100 will probably be sufficient. Express
results so as to indicate per cent, of organisms destroyed by the
process. Compare the keeping qualities of the pasteurized pro-
duct with that of the raw milk by
keeping samples of both under similar
conditions, e. g. in locker or ice chest,
making frequent observations.
Pasteurized milk should not have
a permanently cooked taste.
REFERENCES. H. 485; Wis. Exp,
Station Bull. No. 44 and 18th. An.
Rept. 185. Russell, Outlines of Dairy
FIG. 30. The Freeman Pasteurizer. Bacteriology, (5th Edit.) 113.
EXERCISE 72. TESTING ANTISEPTIC ACTION OF CHEMICALS.
GENERAL DIRECTIONS.
a. Fill a number of test-tubes with a measured quantity of agar
(5 ce).
&. Add to the agar varying but measured amounts of the sub-
stance to be tested. If the antiseptic be not volatile, or affected by
heat, sterilize.
c. Inoculate the tubes thus prepared, together with a control,
with B. coli or M. pyogenes and make rolls.
d. Keep these cultures under observation in the incubator.
e. If no growth appears within 96 hours repeat the experiment,
using smaller amounts of the antiseptic. In this way determine the
amount of chemical (in %) which just prevents growth.
SPECIAL DIRECTIONS. Test in this way carbolic acid (5%),
alcohol (95 %).
REFERENCES. A. 619 ; H. 506 ; M. & R. 140 ; McF. 248.
136 GENERAL BACTERIOLOGY
EXERCISE 73. TESTING DISINFECTING ACTION OF CHEMICALS.
SUSPENSION METHOD.
a. Make a culture of the organism to be studied in tubes of
bouillon containing 5 cc.
&. Incubate at 38° C. for 24 hours.
c. Add to this an equal amount (5 cc.) of the disinfectant to be
tested, of double the required strength.
d. At the end of 5, 10, 20, 40 and 60 minutes make agar rolls,
using two or three loopfuls of the mixture for each roll.
e. In this way determine the time of exposure necessary to kill
the organism used.
/. Test in this way the value of corrosive sublimate (1:1000)
and Lysol (5%), using B. coli or M. pyogenes.vsx. aureus.
COVER-GLASS METHOD.
a. Make a bouillon culture of the organism to be studied and
incubate at 38° C. for 24 hours.
6. By means of a burette, pipette, or loop, place the same sized
drop on each of several sterile cover-glasses and dry as directed in
the experiment on desiccation (43).
c. When the cover-glasses are dry, they are to be immersed in
the disinfectant for the stated periods of time; then removed,
washed in sterile water and transferred to tubes of melted agar
which are then made into rolls.
d. Test by this method carbolic acid (5%), alcohol (95%) and
formaldehyde (4%) or formalin (10%), using B. coli.
REFERENCES. A. 611 ; McF. 249 ; N. 518 ; P. 152 : S. 158.
PLATE I
ooooooooo o
»-<» «n<o'**ww °
Facing page 136>,
O O O O O
to ^ in © r*-
GASOMETER FOB FERMENTATION TUBE.
(See page 72).
PLATE II
100
a.
b.
PLATE COUNTER (Modified from Jeffers).
FOR COUNTING COLONIES OF BACTERIA.
The cross lines divide the figure into square centimeters. The
numbers indicate the area of the various discs. The area of each
sector (a. and b.) is one-tenth of the whole area. (See page 126.)
Pacing page 137.
PART II
MEDICAL BACTERIOLOGY
138 MEDICAL BACTERIOLOGY
PART II-MEDICAL BACTERIOLOGY
CHAPTER VI
PATHOGENIC AEROBES
EXERCISE 74. PREPARATION OF CULTURE MEDIA.
The following media will be necessary for the work outlined in
the following chapters. This is exclusive of a few special media
which are described under special heads and are to be made as a
part of the exercises in which they are used.
100 tubes of agar.
12 tubes of dextrose agar.
100 tubes of gelatin.
12 tubes of dextrose gelatin.
30 tubes of bouillon.
10 fermentation tubes of dextrose bouillon.
35 tubes of potato.
35 tubes of litmus milk.
35 tubes of dextrose free broth or Dunham's solution.
30 water blanks.
30 tubes of Loeffler 's blood serum. This is prepared as follows :
a. Collection of the blood. Sterilize Mason fruit jars, by suc-
cessive washings in corrosive sublimate, distilled water, alcohol and
ether (or a large pail may be used). These are to be carried to
the slaughter house and the blood from a beef caught directly into
them. The blood is then allowed to stand undisturbed for 15 to
30 minutes, or until the clot has firmly attached itself to the sides
of the vessels, when they are to be covered and removed to the
laboratory.
6. Separation of the serum from the blood clot. The clot is
separated from the sides of the vessel by means of a sterile knife or
glass rod, and the vessel placed in the ice chest. After standing
48 hours the clot will have shrunken away from the walls of the
140
MEDICAL BACTERIOLOGY
vessel leaving the clear serum on the top and at the sides. This
can now be pipetted or siphoned off. If the serum contains a large
number of red blood corpuscles it can be placed in rather tall cylin-
ders (graduates) and allowed to stand 24 hours longer, when the
clear straw colored serum can be readily separated. This may be
preserved for a long time by the addition of %% chloroform and
kept in a tightly corked bottle in a cool place.
c. Loeffler's mixture. This consists of 3 parts of blood serum
and 1 part of \% dextrose bouillon.
d. Sterilization. Fill sterile test-tubes (about 3 cm. deep) with
the serum mixture and place them im-
mediately in a sloping position in an
inspissator (Fig. 31), or steamer and
heat to 95° C. for 1 hour on three con-
secutive days. If a higher tempera-
ture be employed bubbles are formed
which rupture the surface of the me-
dium in their escape. When steril-
ized the tubes should be sealed with
paraffin or otherwise.
REFERENCES. A. 110; H. 51; M.
&T? AQ i\/r«Tn 1 on XT
K. 4o ; Me.b . Io7 ; JN.
T>
r.
PIG. 31. Blood Serum Inspissator,
(Muir & Ritchie).
EXERCISE 75. ERYSIPELAS GROUP.
Streptococcus erysipelatos FEHLEISEN.
HI
SYNONYMS. Streptococcus pyogenes ROSENBACH ; streptococcus.
EXPLANATORY. First described by Fehleisen. It is found in
abscesses, pyemia, puerperal fever and erysipelas. It is frequently
present in mixed or secondary infections, and occurs in the mouth
and sputum and on the mucous membranes of the nose, urethra,
vagina, etc.
REFERENCES. Fehleisen, Aetiol. des Erysipels, Berlin 1883 ;
A. 279 ; C. 65 ; H. 165 ; K. & W. Ill, 303 ; L. & N. 135 ; Mig. 2, 6 ;
M. & R. 184 ; McF. 262 ; P. 476.
MORPHOLOGICAL. CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
?> . Agar
c. Gelatin
d. Other media
2. SIZE :
3. STAINING POWERS:
a. Aqueous gentian- violet
6. Loeffler's methylen-blue
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement
6. Flagrella stain
5. SPORES:
6. SPECIAL CHARACTERS: ,
a. Capsules ,
6. Involution forms ,
c. Deposits or vacuoles
d. Pleomorphism
142 CULTURE CHARACTERS
Reaction of media (Fuller's scale) 4. or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at
Agar plate: Grown 24 hours at °C.
(a) Surf ace Colonies. : (b) Deep Colonies.
Sketches.
op
•c
Special Media: (Sucb as liinaus milk and blood serum.)
STREPTOCOCCUS ERYSIPELATOS
143
Gelatin Stab: Grown 24 hours at . °C *••}
Q
0
i
•8
£
2
§
§
J3
V )
A
V )
48 hours at °C.
6 days at C.
Agar Streak: Grown 24 hours at
48 hours at
days at °C.
Potato: Grown 24 hours at
48 hours at . °C.
6 days at..
Bouillon: Grown 24 hours at .. . °C r^
ts
oo
O
v 2
48 hours at »C.
6 days at °C.
144 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE :
optimum °C. ; limits to °C. ;
thermal death-point °C. ; time of exposure minutes ;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION;
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture :
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CO2: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OP NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic...
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 76. PUS COCCUS GROUP.
Micrococcus pyogenes var. albus (EOSENBACH) L. & N.
145
SYNONYMS. Staphylococcits pyogenes albus KOSENBACH;
Staphylococcus epidermidis albus WELCH ; white staphylococcus.
EXPLANATORY. First described by Rosenbach. One of the com-
mon organisms found in pus. Occurs on the skin, in sputum, air,
water, dust and soil.
REFERENCES. Rosenbach, Mikroorganismen bei dem Wundinfec-
tionskrankheiten des Menschen. 1884; C. 75 ; K. & W. Ill, 105;
L. & N. 180 ; Mig. 2, 87 ; McF. 255 : P. 470.
MORPHOLOGICAL CHARACTERS
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
f>. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet. .
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
b. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS :
a. Capsules
b. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
146 CULTURE CHARACTERS
Eeaction of media (Puller's scale) -|_
or —
Gelatin plate: Grown 24 hours at °C.
a) Surface Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Agar plate: Grown 24 hours at °C.
Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
MICROCOCCUS PYOGENES var. ALBUS
147
Gelatin Stab: Grown 24 iiours at*. .......... °C r^
0
?
«
1
O
\ /
"S
\ /
48 hours at
'C.
6 days at 0C.
Agar Streak: Grown 24 hours at ,
48 hours at °C.
Gdaysat °C.
Potato : Grown 24 hours at °C.
48 hours at °C.
6 days at • °C
Bouillon: Grown 24 hours at.
Q
48 hours at °C.
6 days at °C.
148 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C.; limits to °C.;
thermal death-point °C. ; time of exposure minutes ;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3 RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
'gas formula, H: COz: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION :
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) : . ,
PUS COCCUS GROUP— CONTINUED.
149
Micrococcus pyogenes var. aureus (KOSENBACH) L. & N.
SYNONYMS. Staphylococcus pyogenes aureus ROSENBACH ; Golden
pus coccus.
EXPLANATORY. First described in 1884 by Rosenbach. It is the
most common organism in pus— 80%.
REFERENCES. Rosenbach, Mikroorganismen bei dem Wundin-
fectionskrankheiten des Menschen, 1884; A. 270; C. 89; Fl. 2, 96;
H. 162; K. & W. III. 105; L. & N. 180; Mig. 2, 135; M. & R. 182;
McF. 256 ; P. 461.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet. .
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
«. Character of movement. .
6. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
150 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -}_ or —
Gelatin plate: Grown 24 hours at ,...°C.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches
48 hours at.
6 days at.,...,... .»C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches,
48hoursat.
6 days at.
Special Media: (Such as litmus milk and blood serum.)
MICROCOCCUS PYOGENES var. AUREUS
151
Gelatin Stab : Grown 24 hours at °C.
48 hours at °C.
6 days at oC.
Agar Streak: Grown 24 hours at °C.
hours at °C.
6daysat °C
Potato: Grown 24 hours at °C.
48 hours at °C.
6 days at ....°C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at. °C
152
PHYSIOLOGICAL CHARACTERS
lo RELATION TO TEMPERATURE:
optimum. °C. ; limits to °C.:
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN: '.
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :—
4. PIGMENT PRODUCTION :
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm .
rate of development: 24 hours per cent., 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CO2: : :
l». lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia
8. INDOL PRODUCTION
48 hours days
9. ENZYME PRODUCTION:
proteoly tic
digestion of gelatin digestion of casein
diastatic •
10. CHARACTERISTIC ODOK:
11. PATHOGENESIS (or other special characters):.
EXERCISE 77. MALTA FEVER GROUP. 153
Micrococcus melitensis BRUCE.
EXPLANATORY. This organism is the cause of Malta fever and
is found especially in the spleen of the diseased.
REFERENCES. Bruce, Practitioner, Sept. 1887 and Ann. de
1'Inst. Pasteur, 1893, 7, 289; Durham, Jour. Path, and Bact., 1898,
5, 377 ; H. 441 ; K. & W. Ill, 438 ; L. & N. 168 ; Mig. 2, 83 ; McF. 581 ;
M. & R. 452.
MORPHOLOGICAL CHARACTERS:
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
I. Agar
c. Gelatin.
d. Other media..
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet. .
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
b. Flagella stain
5. SPORES:
& SPECIAL CHARACTERS:
a. Capsules
b. Involution forms ,
c. Deposits or vacuoles..
d. Pleomorphism
154 CULTURE CHARACTERS
Reaction of media (Fuller's scale) _j_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies, (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at "C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C. 6 days at
Special Media: (Such as litmus milk and blood seruinj
MICROCOCCUS MELITENSIS
155
Gelatin Stab: Grown24hours at °C.
48 hours at . .
6 days at oC.
Agar Streak: Grown 24 hours at °C.
48 hours at °C.
6 days at.
Potato : Grown 24 hours at °C.
O
48 hours at
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
156 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
«. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours per cent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H: CO2-. : :
?>. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION : - - » , ,
litmus milk
7. REDUCTION OF NITRATES:
to nitrites . - to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) : . ,
EXERCISE 78. DIPLOCOCCUS GROUP. 157
Micrococcus gonorrhoeae (BAUM) FLUEGGE.
SYNONYMS. Gonococcus; Diplococcus gonorrhoeae BAUM.
EXPLANATORY. First described in 1879 by Neisser. It is con-
stantly found in gonorrhoeal discharges and may produce disease
on any mucous membrane; urethra, bladder, rectum, conjunctiva
(causing ophthalmia neonatorum), and even cause arthritis (gonor-
rhoeal rheumatism), endocarditis, salpingitis and general septicemia.
REFERENCES. Neisser, Cent. f. d. Mediz. Wissensch., 1879, 497;
Foulerton, Trans. Brit. Inst. of Prev. Med., 1897, 1, 40 ; A. 288 ;
C. 72 ; H. 179 ; K. & W. Ill, 148 ; L. & N. 164 ; Mig. 2, 188 ; M. & R.
189 ; McF. 275 ; P. 522.
CULTURE CHARACTERS.
MORPHOLOGICAL CHARACTERS:
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin
d. Other media, >
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
&. Loeffler's methylen-blue.
c. Gram's stain »
d. Special stains,
4. MOTILITY:
a. Character of movement..
6. Flagella stain
5. SPORES :
6. SPECIAL, CHARACTERS:
a. Capsules
b. Involution forms «...
c. Deposits or vacuoles
d. Pleomorphism
158 CULTURE CHARACTERS
The Micrococcus gonorrhoeae does not grow on the ordinary arti-
ficial media but may be cultivated on the following :
a. Blood agar. Blood drawn from the finger, under aseptic pre-
cautions, into a capillary pipette is placed on the surface of agar
either in tube or Petri dish. This blood is then inoculated with
the material containing the organism (pus or pure culture) and
smeared over the surface of the agar either with the loop, or better,
by means of a sterile camel's hair brush.
&. Wertheim's method. Human blood-serum (from placenta or
pleuritic or other effusion may be used) in a fluid and sterile condi-
tion is placed in two or three test-tubes. These are heated to 40° C.
and inoculated with the material containing the organism, making
dilutions from one to another, if necessary. To each tube is then
added an equal quantity of nutrient (ordinary or 2%) agar thor-
oughly liquefied and cooled to 40° C. The two are then thoroughly
mixed and quickly poured into Petri dishes and placed in the incu-
bator at 38° C. Colonies appear in 24 hours.
c. Eabbit blood-serum may be used either in a fluid or solid
condition.
DIPLOCOCCUS GROUP— CONTINUED.
159
Micrococcus Weichselbaumii (TREVISAN).
SYNONYM. Diplococcus intracellularis meningitidis WEICHSEL-
BAUM.
EXPLANATORY. First described in 1887 by Weichselbaum. It is
found in the meningeal exudate of certain cases of epidemic cerebro-
spinal meningitis and in nasal secretions in a number of cases.
REFERENCES. Weichselbaum, Fortschritte der Medicin, 1887 ;
Councilman, Kept. Mass. State B. of H. 1898 ; A. 298 ; C. 64 ; H. 170 ;
K. & W. Ill, 256 ; L. & N. 148 ; Mig. 2, 189 ; McF. 281 ; M. & R. 188 ;
P. 516; S. 310.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agar
c. Gelatin
d. Other media.
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet
&. Loeffler's methylen-blue
c. Gram's stain
d. Special stains
4. MOTILIT Y :
a. Character of movement —
&. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS :
a. Capsules
&. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
160 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) _}_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies
Sketches,
48 hours at. °C,
6 days at.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
48 hours at.
6 days at.
Sketches.
Special Media: (Such as litmus inilk and blood serum.)
MICROCOCCUS WEICHSELBAUMII
161
Gelatin Stab: Grown 24 hours at °C.
•i
48 hours at ,
6 days at oC.
Agar Streak: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at »C.
6 days at °<X
Bouillon: Grown 24 hours at °C.
48 hours at... °C.
162 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: ,
optimum °C.; limits to °C.;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COz: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
80 INDOL PRODUCTION
48 hours days.
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic...
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 79. SARCINA GROUP.
163
Sarcina tetragena (GAFF KY) MIG.
SYNONYM. Micrococcus tetragenus GAFFKY.
EXPLANATORY. First described in 1883 by Gaffky. It is found
in phthisical eavities and in sputum, and it occasionally occurs in
mixed infections as abscesses connected with carious teeth, about the
neck, jaws, and middle ear ; rarely elsewhere.
REFERENCES. Gaffky, Langenbeck's Archiv, 1883, 28, 500; A.
326 ; C. 84 ; Fl. 2, 155 ; H. 172 ; L. & N. 171 ; Mig. 2, 225 ; M. & R. 187 ;
M. & W. 133 ; McF. 571 ; P. 472.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
•*
b. A gar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet..
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
b. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:
a. Capsules
6. Involution forms
c. Deposits or vacuoles ,
d. Pleomorphism
164 CULTURE CHARACTERS
Keaction of media (Fuller's scale) -J- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at »C.
Agar plate: Grown 24 hours at °C.
(a) Surf ac* Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at. *C.
Special Media: (Such as litmus milk and blood serum.)
SARCINA TETRAGENA
165
Gelatin Stab: Grown 24 hours at.
48 hours at °C.
6 days at oC.
Agar Streak: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
166 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN: ,
3- RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA: c
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CCb: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic ,
digestion of gelatin digestion of casein .
diastatic...
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 80. ANTHRAX GROUP.
167
Bacterium anthracis (KOCH) MIG.
SYNONYMS. Bacillus anthracis KOCH ; anthrax bacillus.
EXPLANATORY. First described by Robert Koch in 1876. Found
in the blood and tissue in cases of anthrax or splenic fever.
REFERENCES. Koch, Cohn 's Beitraege zur Biologic der Pflanzen,
1876, 2, 277; Chester, Rept. Delaware Exp. Station, July, 1895;
A. 492 ; C. 190 ; Fl. 2, 217 ; H. 184 ; K. & W. II, 1 ; L. & K. 287 ; L. &
N. 307 ; Mig. 2, 280 ; M. & R. 300 ; M. & W. 156 ; McF. 469 ; P. 547 ;
S. 328.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin •
d. Other media
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet. .
b. Loeffler's methylen-blue.
r,. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of! movement..
b. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS :
a. Capsules
b. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
168 CULTURE CHARACTERS
Reaction of media (Fuller's scale) -|_ or —
Gelatin plate: Grown 24 hours at °C. | Sketches.
(a) Surface Colonies. (b) Deep Colonies, j
48 hours at.
«days at
{a) Surf ace Colonies.
Agar plate: Grown 24 hours at °C.
(b) Deep Colonies.
Sketches.
48 hours at •
°C
6 days at . c °C
i
i
i
Special Media: (Such as litmus milk and olood serum.)
BACTERIUM ANTHRACIS
169
Gelatin Stab: Grown 24 hours at °C.
48 hours at CC.
6 days at «,C.
Agar Streak: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at «C.
6 days at °C.
170 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: ..4,
optimum ° C. ; limits to ° C. ;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN: ,
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COz: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic .
rO. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters):.
EXERCISE 81. FRIEDLANDEE GROUP.
171
Bacterium pneumonicum (FRIED.) MIG.
SYNONYMS. Friedlander's bacillus; bacillus pneumonias WEICH-
SELBAUM ; Pneumobacillus of Friedlander.
EXPLANATORY. First described by Friedlander in 1882. Found
frequently in normal saliva, lungs, "rusty sputum " of pneumonia,
and has been found in air and water.
REFERENCES. Friedlander, Virchow's Archiv, 32, 319; C. 131;
Fl. 2, 342 ; H. 314 ; K. & W. Ill, 189 ; L. & N. 225 ; Mig. 2, 350 ; M. &
R. 209 ; McF. 300 ; P. 458.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agrar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian-violet..
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILJTY:
a. Character of movement..
6. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
172 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) 4. or — .
Gelatin plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
48 hours at °C. 6 days at °C.
Agar plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
48 hours at °C. 6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM PNEUMONICUM
173
Gelatin Stab: Grown 24 hours at °C.
48 hours at °C.
6 days at »C.
Agar Streak: Grown 24 hours at °C.
I 6
48 hours at °C.
6 days at °C.
Potato: Grown 24 hours at
9 \
48 hours at °C.
Sdaysat °C.
Bouillon: Grown 24 hours at.
48 hours at °C.
6 days at °C.
174 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C. ; time of exposure minutes ;
medium in which exposure is made
2. RELATION TO FREE OXYGEN :
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc-:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development : 24 hours per cent. . 48 hours per cent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COa: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:...
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
FRIEDLANDER GROUP— CONTINUED.
175
Bacterium aerogenes (ESCH.) Mm.
SYNONYMS. Bacterium lactis aerogenes ESCHERICH; Bacillus
aerogenes KRUSE.
EXPLANATORY. This organism was first described by Escherich,
who isolated it from the milk-stools of infants. It is very similar to
Bact. acidi-lactici, and often difficult to differentiate from B. coli.
Found in milk, feces, air, water, etc.
REFERENCES. Escherich, Fortschritte der Medizin, 1885, No. 16-
17. C. 128 ; L. & N. 221 ; Mig. 2, 396.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin
d. Other-media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
&. Flagella stain
5. SPORES :
6. SPECIAL, CHARACTERS :
a. Capsules
&. Involution forms
c. Deposits or vacuoles
d. Pleomorphism ,.
176 CULTURE CHARACTERS
Reaction of media (Fuller's scale) -j- or —
Gelatin plate: Grown 24 hours at °C.
*a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Agar plate: Grown 24 hours at
°C.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches.
48 hours at
°c
6 days at
°c
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM AEROGENES
177
Gelatin Stab* Grown 24 hours at °C r^
i
s
O
hours at °C
15 hours at . . . , °C. 6 days at . .
Agar Streak: Grown 24 hours at C.
O
O i
48 hours at
Gdaysat... °C.
Potato : Grown 24 hours at °C.
48 hours at °C.
6 days at..
Bouillon: Grown 24 hours at.
dours at
'C.
6 days at. .
178 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN : ,
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.: — .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent., 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H : COa: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk .
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION —
48 hours days ,
9. ENZYME PRODUCTION:
proteolytic -
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) ;
FRIEDLANDER GROUP— CONTINUED.
Bacterium capsulatum (STERNBERG) CHESTER.
179
SYNONYMS. Pfeiffer's capsule bacillus; Bacillus capsulatus
STERNBERG.
EXPLANATORY. First described by Pfeiffer, who isolated it from
the blood of guinea pigs which died spontaneously.
REFERENCES. Pfeiffer, Z. f . H. 1889, 6, 145 ; C. 129 ; L. & N. 228 ;
Mig. 2, 349.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
1. Agar r.
c. Gelatin
d. Other media
••
2. SIZE:
2. STAINING POWERS:
a. Aqueous gentian- violet
6. Loeffler' a tnethylen-blue
c. Gram's stain
d. Special stains
&, MOTILJT Y :
a. Character of movement
6. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS :
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
180 CULTURE CHARACTERS
Reaction of media (Fuller's scale) J_
or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at »C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. : (b) Deep Colonies.
Sketches.
48 hours at • . • •
°C
°c
\.
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM CAPSULATUM
181
Gelatin Stab: Grown 24 hours at °C. Q
48 hours at
C.
6 days at oC.
Agar Streak: Grown 24 hours at
48 hours at
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
182 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. :
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2; Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours per cent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: €62: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION: . .
proteolytic
digestion of erelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters)
EXERCISE 82. SWINE PLAGUE GROUP. 183
Bacterium cholerae (ZOPF) KITT.
SYNONYMS. Bacillus of chicken cholera; Bacillus of swine
plague; Bacterium cholerge-gallinarum CROOKSHANK; Bact. suicida
MIGULA ; Bacillus septicemiae hemorrhagicse HUEPPE.
EXPLANATORY. First described by Koch in 1878. Found in
blood, organs and excreta of chickens suffering with fowl cholera,
and in swine suffering from swine plague.
REFERENCES. Koch, Wundinfectionskrankheiten, Septikaemie
bei Kaninchen, 1878 ; Smith, Report on Swine Plague, Bureau of
Animal Industry, U. S. Dept. Agri., 1891 ; Smith & Moore, Bull. 6,
B. A. I., 1894 ; C. 135 ; H. 305 ; K. & W. II, 543 ; L. & N. 208 ; Mig. 2,
364 ; McF. 534.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
c. Gelatin.
d. Other media.
2. SIZE:
•3. STAINING POWERS:
a. Aqueous gentian-violet..
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
6. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:....
a. Capsules
6. Involution forms —
c. Deposits or vacuoles.
«"'. Pleomorphism
184 CULTURE CHARACTERS
Reaction of media (Fuller's scale) _|_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
°c
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM CHOLERAE
185
Gelatin Stab: Grown 24 hours at °C.
v ./
48 hours at
6 days at
Agar Streak: Growr 24 hours at °C.
48 hours at
6 days at °C.
Potato: Grown 24 hours at
48 hours at °C.
6 days at °C.
Bouillon: Grown 24 hours at °C.
« !
4« hours »t
6 days at.
186 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C.; limits to °C.;
thermal death-point °C. ; time of exposure minutes :
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.: — .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hoars. per cent., 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H : CO2: : :
&. lactose c, saccharose
6. ACID OR ALKALI PRODUCTION : -
litmus milk,
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
80 INDOL PRODUCTION
48 hours. days .
9. ENZYME PRODUCTION: . .
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) : . .
SWINE PLAGUE GROUP— CONTINUED.
187
Bacterium bovisepticum (KRUSE) MIG.
SYNONYMS. Bacillus of hemorrhagic septicemia; Bacillus bovi*
septicus KRUSE.
EXPLANATORY. First described by Bellinger, 1878. It is the
cause of hemorrhagic septicemia in cattle and in other animals.
KEFERENCES. Bellinger, Ueber eine neue Wild und Rinder-
seuche, Muenchen, 1878 ; C. 137 ; K. & W. II, 559 ; Mig. 2, 367.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet..
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement. .
&. Flagella stain
5. SPORES :
6. SPECIAL. CHARACTERS:
a. Capsules
&. Involution forms
c, Deposits or vacuoles
d. Pleomorphism...... .....
188 CULTURE CHARACTERS
"Reaction of media (Fuller's scale) -J_ ....
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
40 uours at °C.
6 days at «C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
»C
«c.
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM BOVISEPTICUM
189
Gelatin Stab: Grown 24 hours at.
48 hours at °C.
6 days at oC.
Agar Streak: Grown 24 hours at °C.
48 hours at
6 days at °C.
Potato: Grown 24 hours at
48 hours at , °C.
6 days at °C.
Bouillon: Grown 24 hours at.
48 hours at °C.
6 days at °C.
190 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: c «,....
optimum °C. ; limits to °C.;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made :
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. : — .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours per cent.
72 hours per cent., .hours per cent.
reaction iu open arm
gas formula, H: COa: : :
fe. lactose « c. saccharose
6. ACID OR ALKALI PRODUCTION : - -
litmus milk.
7. REDUCTION OP NITRATES:
to nitrites to ammonia.. .
8. INDOL PRODUCTION
48 hours days .
8. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic ,
10. CHARACTERISTIC ODOR:.....
11. PATHOGENESIS (or other special characters) :
EXEECISE 83. GLANDERS GROUP.
191
Bacterium mallei (LOEFFLER) MIG.
SYNONYMS. Bacillus mallei LOEFFLER ; Bacillus of Glanders.
EXPLANATORY. First described by Loeffler in 1886. Found in
the nodules, ulcers, discharges, etc., of glanders or farcy.
EEFERENCES. Loeffler, Arbeit, aus dem Kaiserl. Gesundheit-
samte, 1886, 1, 141 : A. 376 ; H. 256 ; K. & W. II, 707 ; L. & K. 300 ;
L. & N. 384 ; Mig. 2, 498 ; M. & R. 275 ; M. & W. 164 ; McF. 359 ; P. 508.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
b. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d. Pleomorphism ,
192 CULTURE CHARACTERS
Reaction of media (Fuller's scale) _|_ or —
Gelatin plate: Grown 24 hours at.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM MALLEI
193
Gelatin Stab: Grown 24 hours at ...°C.
§
i I
48 hours at
3C.
6 days at
Agar Streak: Grown 24 hours at °C. j-j
V
48 hours at °C.
6 days at °C.
Potato: Grown 24 hours at °C.
05
I
48 hours at °C. 6 days at °C.
Bouillon: Grown 24 hours at °C.
•g
' j
\
^ x,
48hoursat °C. I 6 days at..
194 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: ,
optimum ° C. ; limits to °C. j
thermal death-point °C. ; time of exposure minutes ;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COz: : :
ft. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:...
litmus milk ,
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days ,
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
GLANDERS GROUP— CONTINUED.
195
Bacterium rhusiopathiae (KITT.) Mio.
SYNONYMS. Bacillus of swine erysipelas; Bacterium erysipe-
latus-suis MIGULA.
EXPLANATORY. First described by Loeffler in 1882. Found in
blood, internal organs, etc., of swine infected with the disease.
REFERENCES. Loeffler. Arb. aus dem Kaiserl. Gesundheitsamte,
1885, 1, 46 ; C. 352 ; K. & W. Ill, 711 ; L. & N. 300 ; Mig. 2, 431 ;
McF. 552.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agrar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS: o „
a. Aqueous gentian-violet
6. Loeffler's methylen-blne
c. Gram's stain
d. Special stains
4. MOTILJTY : ,
a. Character of movement „
&. Flagella stain
5. SPORES : .
6. SPECIAL CHARACTERS :.,
a. Capsules
b. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
196 CULTURE CHARACTERS
Reaction of media (Fuller's scale) -|_ or —
Gelatin plate: Growa 24 hours at..., ...... °C.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches,
48 hours at »C.
6 days at »C.
Agar plate: Grown 24 hours at.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches.
OQ
6 days at
•c
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM RHUSIOPATHIAE
197
Gelatin Stab: Grown 24 hours at.
4»
48 hours at
6 days at oC.
Agar 5treaK: Grown 24 hours at
°C.
•a
£
48 hours at
6 days at.... °C.
Potato: Grown 24 hours at
48 hours at
6 days at..
Bouillon: Grown 24 hours at °(
48 hours at °C.
6 days at °C.
198 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: •
optimum °C.; limits to °C.;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN :
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:.
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture :
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent., 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COz: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days
9. ENZYME PRODUCTION:
proteolytic •
'digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :.,
EXERCISE 84. DIPHTHERIA GROUP. 199
Bacterium diphtherias (LOEFFLER) MIG.
SYNONYMS. Bacillus diphtherias LOEFFLER; Klebs-Loeffler
bacillus.
EXPLANATORY. First described in 1883 by Klebs. First culti-
vated in 1884 by Loeffler. Found in the false membrane* in cases of
diphtheria, and in small numbers in spleen, liver, etc.; occasionally
in healthy throats.
REFERENCES. Klebs, Verhandl. d. Kongress fuer innere Medi-
zin, 1883? II; Loeffler, Mitth. aus dem Kaiserl. Gesundheitsamte,
1884, 2, 421 ; A. 386 ; C. 354 ; Fl. 2, 460 ; H. 194 ; K. & W. II. 759 ;
L. & K. 207 ; L. & N. 389 ; Mig. 2, 499 ; M. & R. 356 ; M. & W. 137 ;
McF. 410 ; P. 229.
MORPHOLOGICAL, CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet
&. Loeffler's methyien-blue
c. Gram's stain
(/. Special stains
4. MOTILIT Y :
a. Character of movement
&. Flagella stain
5. SPORES :
6. SPECIAL. CHARACTERS :
a. Capsules
&. Involution forms
c. Deposits or vacuoles
200 CULTURE CHARACTERS
Eeaetion of media (Fuller's scale) -J- or —
Gelatin plate: Grown 24 Nhours at °C.
a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Agar plate: Grown 24 hours at °C.
(a) Surf ace Colonies. : (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM DIPHTHERIAE
201
Gelatin Stab: Grown 24 hours at °C. Q
I
48 hours at °C.
6 days at
Agar Streak: Grown 24 hours at °C.
48 hours at °C. 6 days at
Potato: Grown 24 hours at °C. qj
I A. 'I
\ '
/
)
48 hours at °C. 6 day s at °C.
Bouillon: Grown 24 hours at °C.
48 hours at ..°C.
Gdaysat °C.
203 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point , °C.; time of exposure minutes;
medium in which exposure is made. . . ;
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development : 24 hours per cent. . 48 hours per cent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COa: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia.. .
8. INDOL PRODUCTION —
48 hours days .
9. ENZYME PRODUCTION:
proteoly tic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters):..
DIPHTHERIA GROUP— CONTINUED.
203
Bacterium pseudodiphtheriticum (LOEFFLER) MIG.
SYNONYMS. Pseudodipktheria bacillus of LOEFFLER; xerose ba-
cillus Of NEISSER-KUSCHBERT.
EXPLANATORY. Isolated by Hoffman and others, from the
healthy mouth and throat; by Neisser-Kuschbert and others from
xerosis and other affections of the conjunctiva. This may be only a
non-virulent variety of Bact. diphtheriae.
KEFERENCES. A. 401 ; C. 355 ; H. 214 ; K. & W. II, 823 ; L. & N.
404 ; Mig. 2, 503 ; M. & R. 370.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
5 . Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement. .
b. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles,,
d. Pleomorphism ,
204 CULTURE CHARACTERS
Reaction of media (Fuller's scale) -j- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
(a) Surface Colonies.
A gar plate: Grown 24 hours at ............ °C.
(b) Deep Colonies.
Sketches.
°c
°c.
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM PSEUDODIPHTHERITICUM
205
Gelatin Stab: Grown 24 hours at °C.
Agar Streak: Grown 24 hours at °C.
! \
—
48 hours at °C
6 days at oC
48 hours at °C.
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at
'C.
6 days at °C.
Bouillon: Grown 24 hours at °C,
48 hours at . . . ,°C.
6 days at.,
206 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. •,
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H: CO2: : :
&. lactose c. saccharose
S. ACID OR ALKALI PRODUCTION :
litmus milk.
7- REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days ,
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 85. PNEUMONIA GROUP.
207
Bacterium pneumoniae (WEICHSELBAUM) MIG.
SYNONYMS. Fraenkel's pneumococcus ; diplococcus pneumoniae
WEICHSELBAUM ; Streptococcus lanceolatus GAMALEIA.
EXPLANATORY. First described by Sternberg in 1880. Found
in saliva and in the nasal secretion of healthy persons— from 20 to 50
per cent. Usually present in "rusty sputum" of pneumonia
patients.
REFERENCES. Weichselbaum, Wiener Med. Jahrbuecher, 1886 ;
Welch, Johns Hop. Hosp. Bulletin, 1892, 3, 125 ; A. 321 ; C. 63 ;
H. 310 ; K. & W. II, 823 ; L. & N. 143 ; Mig. 2, 347 ; M. & R, 208 ;
McF. 289 ; P. 498.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian-violet..
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
A. MOTILITY :
a. Character of movement..
6. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:
a. Capsules o
l>. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
208 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -f- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at.
Agar plate: Grown 24 hours at.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches.
US hours at °C.
6 days at °C.
Special Media: (Suck as litmus milk and blood serum.)
BACTERIUM PNEUMONIAE
209
Gelatin Stab: Grown 24 hours at °C.
Q
48 hours at
6 days at oC.
Agar Streak: Grown 24 hours at °C.
48 hours at °C.
6 days at
>c.
Potato: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at..
210 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE : ,
optimum °C.; limits to °C.;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:... ..' .
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CCb: : :..
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters)
EXERCISE 86. INFLUENZA GROUP.
211
Bacterium influenzae (PFEIFFER) L. & N.
SYNONYM. Bacillus influenzae PFEIFFER.
EXPLANATORY. First described in 1892 by R. Pfeiffer. Found
in the sputum and in nasal secretions of the diseased.
REFERENCES. Pfeiffer, Z. f . H. 1993, 13 ; 357 ; A. 371 ; C. 351 ;
Fl. 2 ; 434 ; H. 316 ; K. & W. Ill, 359 ; L. & N. 202 ; Mig. 2, 506 ; M. &
R, 430;McF. 574; P. 320.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon ,
&. Agar
c. Gelatin
d. Othermedia
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
6. Flagella stain
5. SPORES : . o
6. SPECIAL, CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
212 CULTURE CHARACTERS
B. influenzae does not grow on the ordinary artificial culture
media, but may be cultivated on agar slopes upon the surfaces of
which blood has been smeared. The blood from man, rabbits,
guinea-pigs and frogs can be used, but that from pigeons is best.
The blood may be obtained from a needle prick and spread over
the medium with a loop. The skin should first be washed with
alcohol and then ether and the first drops should not be used. The
sterility of these tubes should be tested by placing them in an incu-
bator for 24 hours previous to inoculation.
EXERCISE 87. TUBERCLE GROUP,
213
Bacterium tuberculosis (Kocn) MIG.
SYNONYMS. Tubercle bacillus; Bacillus tuberculosis KOCH.
EXPLANATORY. First described by Koch in 1882. Found in
diseased tissues of man and animals and in phthisical sputum.
REFERENCES. Koch, Berlin. Klin. Wochenschr., 1882. 15, 221 ;
Smith, Jour. Exp. Med., 1898, 3, 451 ; A. 330 ; C. 356 ; PI. 2, 481 ;
H. 225 ; K. & W. II, 78 ; L. & K. 251 ; L. & N. 410 ; Mig. 2, 492 ; M. &
R. 236 ; M. & W. 148 ; McF. 305 ; P. 623.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet
b. Loeffler's methylen-blue
c. Gram's stain
d. Special stains
4. MOTILIT Y :
a. Character of movement
b . Flagella stain ,
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
b. Involution forms
c. Deposits or vacuoles
d. Pleomorphism 4
214 CULTURE CHARACTERS
Bact. tuberculosis does not grow upon the ordinary artificial
media, but may be grown upon human and beef blood serum, and
after it has been isolated for some time it may be grown upon bouil-
lon, agar and potato to which 5% of glycerine has been added.
Media which are suitable for the isolation of this organism and at
the same time are easy to prepare are Smith's dog blood serum
(Jour. Exp. Med., 1898, 3, 456), and Dorset's Egg Medium. The
last is the simpler, is very satisfactory, and is prepared as follows :
Perfectly fresh eggs are taken, shell broken at one end and the entire
contents poured into a wide mouthed sterile flask. The yolks are
broken with a sterile platinum wire and 25 cc. of water added to
each four eggs, and then the flask is shaken until the contents are
evenly mixed. The mixture is then strained through sterile cloth
which removes the bubbles and makes a homogeneous medium. Run
into sterile test-tubes, about 10 cc. in each, and incline in a serum
inspissator or oven and heat up to 70° C. until coagulated. This
usually requires four to five hours a day for two days. This is all
of the sterilization usually needed. If heated higher the medium
is hardened quicker but the tubercle germ does not seem to grow so
rapidly. Before inoculating the tubes they should be sealed and
placed in the incubator for several days. Cultures from tubercular
lesions are made by tearing the tubercle out with sterile forceps,
crushed as well as possible with the forceps, transferred to the egg-
slopes with a sterile platinum loop ; leave bits of tissue on medium,
avoid breaking surface of medium. (Amer. Med., 1902, and Bull.
52, Part I., Bureau of Animal Industry, 1904.) The tubercle bac-
terium is very sensitive to temperature variations and should there-
fore be kept at a temperature varying at most only a degree or two
from 38° C. It is also extremely sensitive towards desiccation, and,
for this reason, the cotton plug should be well paraffined, or replaced
by a cork through which a small cotton-plugged glass tube passes,
and the atmosphere of the incubator kept saturated with moisture.
BACTERIUM TUBERCULOSIS 215
216
TUBERCLE GROUP— CONTINUED.
Bacterium tuberculosis var. avium (KRUSE) MIG.
SYNONYMS. Bacillus of fowl tuberculosis; Bacillus tuberculosis
avium KRUSE.
EXPLANATORY. This organism was first separated from B.
tuberculosis by Maffucci, and is probably only a variety of the latter.
It is pathogenic for fowl, but ordinarily not for other animals.
REFERENCES. Maffucci, Z. fur H., 1892, 11, 445 ; C. 356 ; K. &
W. II, 127 ; L. & N. 418 ; Mig. 2, 495.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
l>. Agar
c. Gelatin
d . Other media *
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet. .
6- Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement. .
6. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS :
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d, PleomorDhism
BACTERIUM TUBERCULOSIS var. AVIUM 217
Culture requirements practically the same as for Bact. tuber-
culosis.
218 CULTURE CHARACTERS
EXERCISE 88. COLON GROUP.
Bacillus coli (ESCHERICH) MIG.
SYNONYMS. Bacterium coli commune ESCH. ; Colon bacillus.
EXPLANATORY. Escherich was the first to describe this organism
which is widely known as a common inhabitant of the colon of man,
and of some of the lower animals.
REFERENCES. Escherich, Darmbakt. des Sauglings, Stuttgart,
1886 ; A. 432 ; C. 205 ; H. 282 ; K. & W. II, 334 ; L. & N. 243 ; Mig. 2,
734 ; M. & R. 325 ; McF. 510.
MORPHOLOGICAL CHARACTERS :
! SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet..
5. Loeffier's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILIT Y :
a. Character of movement..
6. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:
a. Capsules
b. Involution forms
c. Deposits or vacuoles.. . .
d. Pleomorphism
CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -[- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48hoursat °C. 6 days at
.'C.
(a) Surface Colonies.
Agar plate: Grown 24 hours at °C.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at CC.
Special Media: (Such a« litmus milk and blood serum.)
BACILLUS COLI
221
Gelatin Stab: Grown 24 hours at °C.
11
48 hours at »C. 6 days at oC.
Agar Streak: Grown 24 hours at °C. ^ Q
til/ iv
os w
?v jl
jyj
48 hours at °C. 6 days at °C.
Potato: Grown 24 hours at °C. y
o
i
1
48 hours at ... 1 °C. 6 days at °C
Bouillon: Grown 24 hours at °C. ^ Q
ts is
£ 2
1 I
48hoursat °C. 6 days at ...°C
222 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: ,
optimum °C.; limits to °C.;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN :
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA: c
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent., 48 hours percent
72 hours per cent hours per cent
reaction in open arm
gas formula, H: CO2: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatie. . .
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
COLON GROUP— CONTINUED.
223
Bacillus enteritidis GAERTNER
EXPLANATORY. Isolated by Gaertner from beef in meat poison-
ing case, very closely related to preceding.
REFERENCES. Gaertner, Correspond, d. allg. Artze Vereins,
Thuringen, 1888 ; C. 207 ; Fl. 2, 375 ; K. & W. II, 639 ; L. & N. 251 ;
Mig. 2, 744 ; M. & K. 331 ; McF. 517.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous grentian- violet..
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
6. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
224 CULTURE CHARACTERS
Reaction of media (Fuller's scale) . .
or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at *C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS ENTERITIDIS
225
Gelatin Stab: Grown 24 hours at °C.
48 hours at
6 days at
Agar Streak: Grown 24 hours at °C.
! I!
48 hours at °C. 6 days at °C.
Potato: Grown 24 hours at °C.
/\
/ MX
£
[
V
i
\
\
48 hours at °C. 6 days at °C.
Bouillon: Grown 24 hours at.
48 hours at .................... °C.
6 days at ....................... °C.
226 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CO2: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk .
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 89. HOG CHOLERA GROUP.
227
Bacillus Salmonii (TREVISAN) CHESTER.
SYNONYMS. Hog-cholera bacillus; B. suipestifer KRUSE; Bact.
cholera-suum LEHM. & NEUM.
EXPLANATORY. First described by Klein, 1884, first cultivated
by Salmon and Smith in 1885. Occurs in blood, organs and intes-
tinal contents of hogs suffering from hog cholera.
REFERENCES. Salmon and Smith, Kept. Bureau Anim. Ind.,
1885-91 ; C. 210 ; H. 281 ; K. & W. Ill, 622 ; L. & N. 252 ; Mig. 2, 759 ;
McF. 538.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
i
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY:
a. Character of movement..
b. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
b. Involution forms
c. Deposits or vacuoles ,
d. Pleomorphism
228 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) 4. . , or — .
Gelatin plate: Grown 24 hours at.... °C. Sketches.
(a) Surface Colonies. : (b) Deep Colonies.
I
48hoursat °C. 6 days at
Agar plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. ; (b) Deep Colonies.
48 hours at °C. 6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS SALMONII
229
Gelatin Stab: Grown 24 hours at.
Agar Streak: Grown 24 hours at °C.
O
48 hours at °C.
6 days at . . .... 0C
48 hours at °C.
6 days at °C.
Potato : Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at °C.
230 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C.;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— ,
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent., 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H: COz: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION: . .
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
HOG CHOLERA GROUP— CONTINUED.
Bacillus icteroides SANARELLI.
331
EXPLANATORY. First described in 1897 by Sanarelli, and claimed
by him to be the cause of yellow fever. Very closely related to
preceding.
REFERENCES. Sanarelli, Ann. d. PInst. Past, 1897; L. & N.
256 ; M. & R. 456 ; McF. 525 ; P. 609.
MORPHOLOGICAL CHARACTERS
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian-violet. .
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
&. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles ,
d . Pleomorphism
232 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) _|_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surf ace Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. ; (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS ICTEROIDES
233
Gelatin Stab: Grown 24hours at °C.
48 hours at
H'.
6 days at 0C.
Agar Streak: Grown 24 hours at °C.
48 hours at
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at
'C.
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at,
234 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: «
optimum °C.; limits to °C.;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. EELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc-:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
. (2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent,
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COz: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION : . . . .......
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days ,
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 90. TYPHOID GROUP.
235
Bacillus typhosus ZOPF.
SYNONYMS. Typhoid bacillus; Eberth's bacillus ; Bacillus typhi
ibdominalis AUT.
EXPLANATORY. First described by Eberth in 1880, first culti-
vated by Gaffky, 1884. It is found in the feces and urine of typhoid
patients.
REFERENCES. Eberth, Virchow's Archiv. 1880, 81, 58 and 1881,
83, 486 ; Gaffky, Mitth. aus dem Kaiserlichen Gesundheitsamte, 1884,
2, 372 ; A. 408 ; C. 213 ; H. 263 ; K. & W. II, 204, 166 ; L. & N. 232 ;
Mig. 727 ; M. & R. 319 ; McF. 481 ; P. 402.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
T. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar
c. Gelatin
d. Othermedia
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian-violet. .
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
I. MOTILITY :
a. Character of movement..
b. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
236 CULTURE CHARACTERS
Reaction of media (Fuller's scale) -j- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies . (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
(a) Surface Colonies.
Agar plate: Grown 24 hours at °C.
: (b) Deep Colonies.
Sketches.
48 hours at
oC
«c.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS TYPHOSUS
237
Gelatin Stab: Grown 24 hours at °C.
Agar Streak; Grown 24 hours at
v
6 days at oC
•C.
48 hours at
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at . .
6 days at °C.
Bouillon: Grown 24 hours at.
V
48 hours at °C.
6 days at. ,
238 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C.;
thermal death-point °C. ; time of exposure .minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, ligrht, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5- GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent,
reaction in open arm
gas formula, H: COz: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION :
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
TYPHOID GROUP— CONTINUED. 239
Bacillus dysenteriae SHIGA.
SYNONYM. Bacillus of Japanese dysentery, SHIGA.
EXPLANATORY, First described by Shiga, who found it causally
related to a dysentery epidemic ; Flexner has more recently found it
in the Philippines and elsewhere.
REFERENCES. Shiga, C. f . B., 1898, 23 ; 599 and 24 : 817, 870 and
913 ; Eldridge, Public Health Repts., 1900, 15 ; p. 1, Flexner, Phil.
Med. Jour. 1900, Sept. 1 ; A. 440 ; C. 228. K. & W. II, 317 ; M. & R.
350 ; McF. 519.
MORPHOLOGICAL CHARACTERS: i SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar..
. Gelatin.
d. Other media..
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement. .
6. Flagella stain
SPORES:
f. SPECIAL CHARACTERS: —
a. Capsules
&. Involution forms
c. Deposits or vacuoles.
d. Pleomorphism
240 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -J- or —
Gelatin plate: Grown 24 hours at °C.
(a,) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at »0.
Agar plate: Grown 24 hours at °C.
t) Surf ace Colonies. (b) Deep Colonu
Sketches.
48 hours at
°C
6 days at
Special Media: (Such as litmus milk and blood serum.)
BACILLUS DYSENTERIAE
241
Gelatin Stab: Grown 24 hours at.*. °C.
! -
48 hours at
6 days at oC.
Agar Streak: Grown 24 hours at °C.
48 hours at . .
6 days at °C.
Potato: Grown 24 hours at
48 hours at °C
6 days at °C
Bouillon: Grown 24 hours at °C.
48 hours »t -C.
6 days at °C,
242 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: « „.
optimum °C. ; limits to °C. ;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:.
5. GAS PRODUCTION IN SUGAR MEDIA: ,
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent., 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H : CC«2: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION : . . .
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein,
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters ):.,
TYPHOID GROUP— CONTINUED.
Bacillus pestis (LEHM. & NEUM.) CHESTER.
S43
SYNONYMS. Bacterium pestis LEHM. & NEUM. ; Bacillus pestis-
bubonicae KRUSE ; Bacillus of bubonic plague.
EXPLANATORY. Described at about the same time independently
by Kitasato and Yersin in 1894. Found in the buboes, and occa-
sionally in the feces, urine and blood and, in the pneumonic form,
in the sputum.
REFERENCES. Kitasato, Lancet, 1894, 2, 428 ; Yersin, Ann. Inst.
Past., 1894, 8, 662 ; A. 310 ; C. 215 ; H. 291 ; K. & W. II, 475 ; M. & R.
435 ; L. & N. 213 ; Mig. 2, 749 ; McF. 559 ; P. 606.
MORPHOLOGICAL CHARACTERS: SKETCHES
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agar
c. Gelatin
a. Other media
2. SIZE :
3. STAINING POWERS :
a. Aqueous gentian- violet
b. Loeffler's methylen-blue
c. Gram's stain
d. Special stains
4. MOTILIT Y :
a. Character of movement
&. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS : ,
a. Capsules
6. Involution forms
c. Deposits or vacuoles
<l. Pleomorphism
244 CULTURE CHARACTERS
Reaction of media (Fuller 's scale) -|_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at »C.
Agar plate: Grown 24 hours at , ...°C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at ° C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS PESTIS
245
Gelatin Stab: Grown 24hours at °C.
48 hours at .................... °C.
6 days at 0C.
Agar Streak: Grown 24 hours at ................ °C.
48 hours at °C. 6 days at
Potato: Grown 24 hours at °C.
C3
48 hours at °C. 6 days at °C.
Bouillon: Grown 24 hours at °C.
05
2
48 hours at . . . . °C. 6 days at... ...«C.
24G PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN : . . .
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5- GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CO2: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:... ......
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION: ,
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 91. PSEUDOMONAS GROUP.
247
Pseudomonas aeruginosa (SCHROETER) MIG.
SYNONYMS. Bacillus pyocyaneus GESSARD; Bacillus of blue-
green pus.
EXPLANATORY. First described in 1872 by Schroeter. Found
in green pus, and widely distributed in nature.
REFERENCES. Schroeter, Cohn's Beitraege zur Biologie, 1872, 1>
126; Barker, Jour. Am. Med. Asso., 1897, July 31; Jordan, Jour.
Exp. Med., 1890, 627 ; Lartigau, IMd., 1898, 595 ; A. 304 ; C. 321 ;
H. 171 ; K. & W. Ill, 471 ; L. & N. 281 ; Mig. 884 ; M. & R. 186 ; M. &
W. 160 ; McF. 269 ; P. 535 ; S. 454.
MORPHOLOGICAL CHARACTERS-. SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agar
c. Gelatin
d. Other media
2. SIZE :
3. STAINING POWE.RS :
a. Aqueous gentian- violet
b. Loeffler's methylen-blue
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement
b. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS :
a. Capsules
b. Involution forms
c. Deposits or vacuoles
d. Pleomorphism •
248 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -|- or — .
Gelatin plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
48hoursat °C. 6 days at CC.
Agar plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
48 hours at °C. 6 days at
Special Media: (Such as litmus milk and blood serum.)
PSEUDOMONAS AERUGINOSA
249
Gelatin Stab: Grown 24 hours at °C.
4S hours at °C.
6 days at «C.
Agar Streak: Grown 24 hours at
Q
48 hours at
6 days at..
Potato: Grown 24 hours at ,
48 hours at .
6 days at..
Bouillon: Grown 24 hours at.
48 hours at °C.
6 days at.
250 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C.; limits to °C.;
thermal death-point °C.; time of exposure minute*;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS...
desiccation, light, disinfectants, etc.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CO2: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION: ,
litmus milk,
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteoly tic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 92. CHOLERA GROUP.
251
Microspira comma (KOCH) SCHROETER.
SYNONYMS. Spirillum cholerae-asiaticae ZOPF ; Vibrio cholerae
LEHM. & NEUM. ; Comma bacillus ; Cholera bacillus.
EXPLANATORY. First described by Koch in 1884. Found in
the intestinal contents of cholera patients. It has also been isolated
several times from water supplies.
REFERENCES. Koch, Berl. Klin. Wochenschr., 1884, no. 31 u. 32 ;
A. 446 ; C. 335 ; Fl. 2, 527 ; H. 333 ; K. & W. Ill, 1 ; L. & N. 353 ; Mig.
2, 960 ; M. & R. 407 ; M. & W. 152 ; McF. 442 ; P. 568.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet
b. Loeffler's methylen-blue ,
c. Gram's stain ,
d. Special stains ,
4. MOTILITY :
a. Character of movement
b. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS :
a. Capsules
&. Involution forms >
c. Deposits or vacuoles
d. Pleomorphism
252 CULTURE CHARACTERS
Reaction of media (Fuller 's scale) _}_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surf ace Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at »C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
MICROSPIRA COMMA
253
Gelatin Stab : Grown 24 hours at °C.
o
48 hours at °C. 6 days at
Agar Streak: Grown 24 hours at °C. Q p
• _
"i \i
\
\
48 hours at °C. 6 days at °C
Potato: Grown 24 hours at °C. p o
^X 47 ^
c
48 hours at °C. 6 days at °C.
Bouillon: Grown 24 hours at °C. rj Q
V
48 hours at °C.
6 days at °C.
254 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, ligrht, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent., 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CO2-. : : „
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteoly tic
digestion of gelatin digestion of casein.
diastatic •
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
CHOLERA GROUP— CONTINUED.
255
Microspira Metschnikovi (GAMALEIA) MIG.
SYNONYM. Vibrio Metschnikovi GAMALEIA.
EXPLANATORY. First described in 1888 by Gamaleia. Fonnd
in intestinal contents, in blood and in organs of chickens suffering
from a disease resembling chicken cholera.
REFERENCES. Gamaleia, Ann. Inst. Past., 1888, 2, 482 ; A. 485 ;
C. 334 ; H. 345 ; K. & W. Ill, 68 ; L. & N. 366 ; Mig. 2, 979 ; M. & K.
427 ; McF. 462 ; P. 593.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY: ,
a. Character of movement..
b. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
b. Involution forms
c. Deposits or vacuoles ,
d. Pleomorphism
256 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) _|_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
•
0£«
«c
A gar plate: Grown 24 hours at.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6days at °C.
Special Media: (Such as litmus milk and blood serum.)
MICROSPIRA METSCHNIKOVI
257
Gelatin Stab: Grown 24 hours at.
SC.
48 hours at
6 days at
Agar Streak.: Grown 24 hours at
48 hours at °C.
6 days at.
Potato: Grown 24 hours at
Bouillon: Grown 24 hours at.
48 hours at °C
6 days at °C
48 hours at °C.
6 days at..
258 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. limits to °C. ;
thermal death-point °C.; timeof exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN: ,
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: ?4 hours per cent., 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CC»2: : :
1. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION : ,
litmus milk.
7. REDUCTION OP NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) : . ,
CHOLERA GROUP — CONTINUED.
Microspira Schuylkilliensis (ABBOTT) CHESTER.
259
SYNONYM. Vibrio Schuylkilliensis ABBOTT.
EXPLANATORY. Isolated from the Schuylkill river water by
Abbott in 1896. Very similar to preceding.
REFERENCES. Abbott, Jour. Exp. Med., 1896, 1, p. 419 ; A. 490 ;
C. 334 ; M. & R. 428 ; McF. 465.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar
c. Gelatin
d . Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian-violet. .
ft. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
&. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS :
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
260 CULTURE CHARACTERS
Keaction of media (Fuller's scale) --
Gelatin plate: Grown 24 hours at °C j Sketches
(a) Surface Colonies.
(b) Deep Colonies, j
48 hours at °C.
6 days at "C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
4b hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
MlCROSPIRA SCHUYLKILLIENSIS
261
Gelatin Stab: Grown 24 hours at °C.
48 hours at . .
6 days at 0C.
Agar Streak: Grown 24 hours at ,
O
48 hours at
6 days at °C.
Potato: Grown 24 hours at °C.
48 hours at
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C. 6 days at °C.
262 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE :
optimum °C. ; limits to °C.;
thermal death-point °C. ; time of exposure minutes ;
medium in which exposure is made
2. RELATION TO FKEB OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COa: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk .
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein,
diastatic ...
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :.
EXERCISE 93. STREPTOTHRIX GROUP.
263
Streptothrix bovis (HARZ) CHESTER.
SYNONYMS. Actinomyces bovis HARZ; Streptothrix actino-
myces Rossi-DoRiA; Oospora bovis SAUV. et RADAIS; ray fungus;
actinomyces.
EXPLANATORY. First described by Bellinger. It occurs in
actinomycosis or lumpy- jaw in cattle, hogs, horses and man. It
probably leads a saprophytic life on plants, etc.
REFERENCES. A. 361 ; C. 361 ; H. 349 ; K. & W. II, 861 ; L. & N.
440 ; M. & R. 287 ; McF. 371.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet..
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY:..
a. Character of movement..
6. Flagella stain
5. SPORES :
<5. SPECIAL CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles
d. Pleomorphism ,
264 CULTURE CHARACTERS
Reaction of media (Fuller 's scale) 4. or —
Gelatin plate: Grown 24 hours at °C. j Sketches.
(a) Surface Colonies. (b) Deep Colonies.
48 hours at °C.
6 days at *C
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
STREPTOTHRIX Bovis
265
Gelatin Stab: Grown 24 hours at °C.
•iS hours at
days at .
Agar Streak: Grown 24 hours at °C.
6 days at..
Potato: Grown 24 hours at
C.
48 hou rs at
°C. 6 days at ....................... °C.
I
Bouillon: Grown 24 hours at °C r^
ta
h
J3
O
«S
O
~^_J
^J
48 hours at °C. 6 days at...
0
n.
266 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. •
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:....
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:.
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CO2: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :.
STREPTOTHRIX GROUP— CONTINUED.
267
Streptothrix Madurae VINCENT.
EXPLANATORY. First described by Vincent. Associated with a
warty, ulcerative affection of the feet, but rarely of the hands.
REFERENCES. Vincent, Ann. Past. Inst., 1894; A. 365; C. 368;
H. 356 ; K. & W. II, 839, III, 454 ; L. & N. 452 ; M. & R. 297 ; McF.
378.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar
c. Gelatin
d. Othermedia
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY:
a. Character of movement..
b. Flagella stain
5. SPORES :
6. SPECIAL, CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles
(1. Pleomorphism
268 CULTURE CHARACTERS
Keaction of media (Fuller's scale) -|- or — .
Gelatin plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
48hoursat °C. 6days at «C.
Agar plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
4
48 hours at °C. 6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
STREPTOTHRIX MADURAE
269
Gelatin Stab* Grown 24 hours at • • °C r^
O
0
ts
"S
o
0
V /
•
V j
48 hours at
6 days at
Agar Streak: Grown 24 hours at ,
I
48 hours at
6 days at.
Potato: Grown 24 hours at
48 hours at
6 days at..
Bouillon: Grown 24 hours at.
48 hours at "C.
6 days at.
270 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum ° C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent., 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H: COz: : :
b, lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION: . .
proteolytic
•
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
MEDICAL BACTERIOLOGY
271
CHAPTER VII
PATHOGENIC ANAEROBES
Anaerobic bacteria may be furnished conditions which permit of
their development, in a variety of ways, and a very considerable
number of pieces of apparatus have been devised to secure this end.
In a general way all of the methods may be grouped under the
following heads :
1. Displacement of air.
2. Absorption of oxygen.
3. Exhaustion of air.
4. Exclusion of air.
5. Miscellaneous methods, in the presence of reducing substances
as litmus, or a strongly aerobic germ, etc.
The first two methods are the most reliable. In the displace-
ment method, hydrogen, carbon dioxide or illuminating gas may
be used; hydrogen is best. This gas is readily prepared by the
action of sulphuric acid (1:8)
on zinc. Either a Kipp gen-
erator may be used or one of
a simpler construction (Fig.
32). The gas should be
washed, 1st, in lead nitrate
to absorb the sulphuretted
hydrogen, 2d, in silver sul-
phate to absorb any arseniur-
etted or phosphuretted hydro-
gen, and 3d, in potassium hy-
drate to remove sulphur and
carbon dioxide.
The cultures should be made in dextrose media (which should
preferably be freshly prepared and always boiled immediately be-
fore being inoculated), either as test-tube or plate cultures. Novy's
anaerobic jars are perhaps the most satisfactory receptacles for the
cultures. (For description of same, see N. 306.)
FIG. 32. Hydrogen Generator.
272
MEDICAL BACTERIOLOGY
In the second method (Buchner's method) an alkaline solution
of pyrogallic acid is used to absorb the oxygen. The cultures may
be placed in Novy jars or similar receptacles; for tube cultures a
large wide mouthed bottle fitted with a rubber cork does very well.
The dry pyrogallic acid is placed in the bottom of the receptacles,
about 1 gram to every 100 cc. of air space, the tubes are put in place,
then about 10 cc. of a normal sodium hydroxide solution are added
to each gram of pyrogallic acid, and the apparatus immediately and
hermetically sealed. A very convenient method has recently been
outlined by Wright for test-tube cultures. It is as follows : After
the culture medium has been inoculated in
the usual manner, thrust the cotton plug
into the test-tube so that the upper end of
the cotton is about 2 cm. below the mouth
of the test-tube (it is usually desirable to
cut off a part of the protruding portion
before doing this). Fill the tube with py-
rogallic acid. Add with a pipette enough
of a 4% solution of sodium hydrate to dis-
solve the acid. Close the tube immediately,
making it air tight by inserting a rubber
stopper in its mouth. Then invert, in the
case of solid media, and set aside for devel-
opment. Fig. 33. Rickards has recently
published a modification, which consists in
inverting the inoculated tubes, without the
plugs, into a glass in which is a layer of dry
pyrogallol and then adding the hydroxide. Plate cultures are made
by using Erlenmeyer flasks instead of Petri dishes.
REFERENCES. A. 206 ; L. & K. 98 ; M. & R. 68 ; M. & W. 117 ;
McF. 216 -P. 233 ; S. 78 ; Wright, Jour. Boston Soc. of Med. Sci.,
1900, 5, 114 ; Rickards, C. f . B., 1st Abt, Originale, 36 ; 557.
FIG. 33. Wright's method
for cultivating anaerobes,
a, cotton plug; b, alkaline
pyrogallic acid solution; c,
rubber cork.
EXERCISE 94. EMPHYSEMA GROUP. 273
Bacterium Welchii MIG.
SYNONYMS. Bacillus aerogenes capsulaius WELCH; B. der Gasphlegmon
FRAENKEL; B. emphyseraatasus KRUSE.
EXPLANATORY. First described by Welch in 1892. Occurs at autopsies in.
which gas bubbles are present in the larger vessels, accompanied by the for-
mation of numerous small cavities in the liver containing gas. It has been
found also in emphysematous phlegmons, in puerperal sepsis, in peritonitis
and in other conditions (M. & W.). Widely distributed in nature. (Welch.)
REFERENCES. Welch and Nuttall, Bull. Johns Hopkins Hospital, 1892, 3,
81; Welch and Flexner, Jour. Exp. Med., 1896, 1, 5; C. 183; H. 329; L. & N.
344; Mig. 392; M. & R. 402; McF. 591; P. 545.
MORPHOLOGICAL CHARACTERS: j SKETCHES.
1. FORM AND ARRANGEMENT:
«. Bouillon :
b. Agrar
c. Gelatin
L Other media.
SIZE:
STAINING POWERS :
a. Aqueous gentian-violet..
b. Loeffler's methylen-blue.
\
c. Gram's stain
d. Special stains
4. MOTILIT Y :
a. Character of movement.
b. Flagella stain
5. SPORES:...
SPECIAL CHARACTERS:....
a. Capsules
b. Involution forms
o. Deposits or vacuoles.
d. Pleomorphism
274 CULTURE CHARACTERS
Eeaction of media (Fuller >s scale) -|_ or —
Gelatin plate: Grown 24 hours at .°C.
(a) Surf ace Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C. 6 days at »C.
Agar plate: Grown 24 hours at °C.
(a) Surf ace Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACTERIUM WELCHII
275
Gelatin Stab: Grown 24 hours at °C.
15
48 hours at °C
6 days at oC
Agar StreaK: Grown 24 hours at °C. rj
48 hours at
6 days at °C.
Potato: Grown 24 hours at °C.
2 i
48 hours at
6 days at.,
Bouillon: Grown 24 hours at.
48 hours at «C. 6 days at..
'C.
276 PHYSIOLOGICAL CHARACTERS
1 RELATION TO TEMPERATURE:
optimum °C. • limits to °C. ;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN :
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, ligrht, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture: c.
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H: CCb: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION: .
litmus milk,
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic «
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :.
EXERCISE 95. OEDEMA GROUP.
277
Bacillus Feseri (TREVISAIN) KITT.
SYNONYMS. Bacillus of symptomatic anthrax; Black-leg bacil-
lus; Bacillus carbonis MIG. ; Bacillus anthracis-symptomatici KRUSE.
EXPLANATORY. First described by Arloing, Cornevin and
Thomas in 1880. It occurs in the subcutaneous tissue, muscles and
serous exudate of animals suffering from symptomatic anthrax.
KEFERENCES. Arloing, Cornevin and Thomas, Le Charbon
symptomatique du boeuf , 2nd edit. Paris, 1887 ; A. 527 ; C. 296 ; Fl.
2, 245 ; H. 328 ; K. & W. II, 601 ; L. & N. 339 ; Mig. 593 ; M. & R. 401 ;
McF. 583 ; P. 563.
MORPHOLOGICAL CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agar
c. Gelatin
d. Other media
2. SIZE :
3. STAINING POWERS :
a. Aqueous gentian- violet
ft. Loeffler's methylen-blue
c. Gram's stain
d. Special stains
4. MOTILIT Y :
a. Character of movement , ,
b. Flagella stain
6. SPORES :
6. SPECIAL CHARACTERS :
a. Capsules
6. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
278 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) _|_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies, (b) Deep Colonies.
Sketches.
48 hours at.
°C. 6 days at.
•C.
Agar plate: Grown 24 hours at °C.
(a) Surf ace Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at 'C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS FESERI
279
Gelatin Stab: Grown 24 hours at °C.
48 hours at °C.
6 days at
Agar Streak: Grown 24 hours at °C. H I
I \.
V
u I
48 hours at
6 days at °C.
Potato : Grown 24 hours at °C.
6 days at °C.
Bouillon: Grown 24 hours at.
48 hours at . .
O
6 days at °C.
280 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum ° C. ; limits to °C.;
thermal death-point °C. ; time of exposure .minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture: ;
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent hours percent.
reaction in open arm
gas formula, H: COz: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION : . . .
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days ,
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :,
OEDEMA GrROOP — CONTINUED.
281
Bacillus oedematis ZOPF.
SYNONYMS. Bacillus of malignant oedema; Bacillus oedematis-
maligni ZOPF.
EXPLANATORY. First described by Pasteur in 1877. Widely
distributed in soil and putrefying material. Few cases on record
of infection in man.
REFERENCES. Zopf, Spaltpilze, 1885, 88; A. 522; C. 292; Fl. 2,
234 ; H. 326 ; K. & W. II, 619 ; L. & N. 341 ; Mig. 604 ; M. & R. 393 ;
Mc.F. 587 ; P. 543 ; S. 488.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
«. Bouillon ,
&. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet..
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
b. Flagella stain
5. SPORES :
6. SPECIAL CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles
d. Pleomorphism
282 CULTURE CHARACTERS
Reaction of media (Fuller's scale) _|_ or — .
Gelatin plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
48 hours at °C. 6 days at , ...,o....*C.
Agar plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies. (b) Deep Colonies.
48 hours at. *C.
6 days at. «C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS OEDEMATIS
283
Gelatin Stab: Grown 24 hours at °C.
Agar Streak: Grown 24 hours at °C.
Potato: Grown 24 hours at °C.
Bouillon: Grown 24 hours at.
48 hours at °C.
6 day*
is
e
6 days at . oC
48 hours at °C
6 days at °C
'C.
284 PHYSIOLOGICAL CHARACTERS
1« RELATION TO TEMPERATURE: ,
optimum °C ; limits to °C.;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.: — .
4. PIGMENT PRODUCTION:
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours .per cent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CO2: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:... .
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:... . .
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
OEDEMA GROUP — CONTINUED.
Bacillus footulinus v. ERMENGEM.
285
EXPLANATORY. Isolated by v. Ermengem from ham which had
caused meat poisoning. Believed to be the cause of meat poisoning
characterized by nervous symptoms of central origin, botulism.
REFERENCES, v. Ermengem, Z. f . H., 1898, 26, 1 ; C. 297 ; K. &
W. II, 671 ; L. & N. 337 ; Mig. 616 ; M. & R. 398.
MORPHOLOGICAL, CHARACTERS: SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon..
6. Agar
c. Gelatin
d. Other media.
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian-violet
6. Loeffler's methylen-blue
c. Gram's stain
d. Special stains
4. MOTILITY:
a. Character of movement. *
6. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:
a. Capsules
b. Involution forms ,..,,
c. Deposits or vacuoles... i •••••••••••••••« ....ft
d. Pleomorwhism..... , ..,...»... ^.......,................. ,,
286 CULTURE CHARACTERS
Reaction of media (Fuller's scale) _j_ or
Gelatin plate: Grown 24 hours at °C. Sketches.
(a) Surface Colonies, (b) Deep Colonies.
48 hours at °C.
6 days at.
(a) Surf ace Colonies.
Agar plate: Grown 24 hours at °C.
: (b) Deep Colonies.
•C.
Sketches.
48 hours at °C.
6days at °C.
Special Media: (Such as litmus milk and blood serum.)
BACILLUS BOTULINUS
287
Gelatin Stab: Grown 24 hours at °C.
Agar Streak: Grown 24 hours at
48 hours at °C
6 days at 0C
o
48 hours at
6 days at..
Potato: Grown 24 hours at
ts y
o
48 hours at .
6 days at °C.
Bouillon: Grown 24 hours at °C.
48 hours at °C.
6 days at. ,
288 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE :
optimum °C. ; limits to °C.;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc : — .
4. PIGMENT PRODUCTION;
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm. . • closed arm
rate of de^7elopment: 24 hours per cent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CC-2: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION: ,
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION: .
proteolytic
digestion of gelatin digestion of casein ,
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
EXERCISE 96. TETANUS GROUP.
289
Bacillus tetani NICOLAIER.
EXPLANATORY. Discovered by Nicolaier, 1884. First cultivated
by Kitasato, 1889. Occurs in man and in animals suffering from
the disease, and is widely distributed in nature, especially in soil.
KEFERENCES. Nicolaier, Deutsche Med. Wochenschrift, 1884;
Kitasato, Deutsche Med. Wochenschrift, 1889 ; A. 513 ; C. 302 ; Fl. 2,
260 ; H. 320 ; K. & W. II, 566 ; L. & N. 332 ; Mig. 592 ; M. & R. 376 ;
McF. 389 ; P. 385.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar
c. Gelatin
d. Other media
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian- violet. .
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains —
4 . MOTILJTY :
a. Character of movement..
6. Flagella stain. .
5. SPORES :...- = ,
6. SPECIAL CHARACTERS:
a. Capsules
&. Involution forms. ........
c. Deposits or vacuoles ,
d. Pleomorphism
290 CULTURE CHARACTERS
Keaction of media (Fuller's scale) _|_ or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. i (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at "C.
Agar plate: Grown 24 hours at.
(a) Surface Colonies.
: (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.;
BACILLUS TETANI
291
Gelatin Stab: Grown 24 hours at °C.
Agar Streak: Grown 24 hours at ,
48 hours at 'C
6 days at 0C
48 hours at .
6 days at °C.
Potato: Grown 24 hours at ,
Bouillon: Grown 24 hours at.
48 hours at °C
6 days at °C
48 hours at °C.
6 days at.,
292 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN :
RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc-:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA :
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent., 48 hours percent.
72 hours per cent hours percent.
reaction in open arm
gas formula, H: COa: : :
b. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:...
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia. .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGEXESIS (or other special characters)
294 MEDICAL BACTERIOLOGY
CHAPTER VIII
ANIMAL INOCULATION AND STAINING OF
BACTERIA IN TISSUE
EXERCISE 97. ANIMAL INOCULATION.
METHODS OF INOCULATION. Animal inoculation is practiced to
determine the pathogenic properties of an organism and also the
character of the tissue changes produced. The animals commonly
used are white mice and white rats, rabbits, guinea pigs and pigeons.
Inoculations are usually made subcutaneously, intraperitoneally
or intravenously, and in special cases into the pleural cavity, brain,
eye, etc., etc. Mice require a holder, the inoculation being usually
made at the root of the tail. Other animals can usually be held by
an assistant.
Subcutaneous. The place selected is usually the abdominal wall.
Pigeons are inoculated in the pectoral muscles ; the hair or feathers
should be removed and the skin washed with a disinfectant, e. g., 5%
carbolic acid.
a. For liquids a sterilized hypodermic syringe is used. A fold
of the skin is raised, the needle of the syringe inserted and the
requisite amount of material injected.
&. For solid material a pocket is made which is stitched, or sealed
with contractile collodion, after the material is introduced.
Intraperitoneal. Either liquids or solid material may be intro-
duced.
a. For liquids. The seat of inoculation is prepared as above, the
syringe needle is then plunged directly into the peritoneal cavity.
6. For solid material. The animal is anesthetized ; the hair is
clipped or shaved from a portion of the median line, about half way
between the pubis and the sternum ; a slit is made in the skin with
sterile instruments ; the smallest possible opening is made along the
linea alba into the peritoneal cavity and the material introduced;
the wound closed and the body wall and the skin stitched separately.
It is hardly necessary to add that the whole operation is carried out
under the most strict aseptic precautions. Collodion sacs are intro-
duced in this way.
296
MEDICAL BACTERIOLOGY
Collodion Sacs. The use of the collodion sac has recently become
very common and deserves description as one of the necessary labor-
atory procedures. The difficulty in making these sacs has been
largely overcome by recent methods. One of these is the following :
Small-sized test-tubes are selected. Thick collodion is then poured
into the tube to a depth of two inches. The collodion is then
poured out along one side of the tube into another tube and from this
one to another and so on until the required number is obtained. The
desired length of the sac can be secured in all of the tubes by tipping
and rolling them, thus bringing the collodion into contact with the
glass to the proper height. As the tubes are coated they are placed,
mouth down, in a wire basket or test-tube rack as indicated in Fig.
34, 1 ) . In this way the extra collodion drains off and free access of
air dries and hardens the collodion, leaving a thin coat covering the
inner surface of the tube. The thickness of the coat depends on the
consistency of the collodion. A ten per cent, collodion, in equal
parts of alcohol and ether, makes a sufficiently thick coat for ordi-
nary purposes. The drying may be stopped at any point by filling
i
Fig. 34. Preparation of Collodion Sac: 1, Tube inverted to allow the extra collodion
to drain off and the film to air-dry. 2, Sac ready for sterilization; (A) Surgeon's knot;
(B) Ends of cord; (C) Tongue of collodion. 3, Sac ready to be inoculated into animal.
the tube with water and after standing a few minutes the collodion
shrinks and the sac may be easily removed. The sacs are then filled
from one-fourth to three-fourths full with bouillon. They are then
immersed in a test-tube of the medium,. The sacs are held in posi-
tion in the test-tube by means of the tongue formed by the collodion
flowing out of the tube. This tongue is folded over the lip of the
tube. (Fig. 34, 2, C.) Before, however, the sac is put into the test-
tube a piece of cotton or silk cord is placed around the sac near the
MEDICAL BACTERIOLOGY
top and held in position by means of a surgeon 's knot, loosely drawn.
The cord should be quite stout so that the sac can later be tightly
closed. The ends of the cord are brought outside of the tube as
shown at B, Fig. 34, 2. Sterilization may be accomplished either in
the autoclave or by means of the intermittent method of sterilization.
The medium is inoculated by means of the platinum needles in
exactly the same way in which tube cultures are ordinarily inocu-
lated. The sac thus inoculated should be incubated for twenty-
four hours and if the medium outside of the sac remains clear the sac
may be used. Otherwise it would be discarded.
The tube is placed in a tumbler or test-tube rack. The' sac is
then pulled out of the tube until the cords can be drawn tight so as
to close the sac and securely tied. With sterile scissors the end of
the sac is cut off a few millimeters above the constriction. If there
is any moisture on the inside of the sac above the neck this must be
removed with sterile filter paper and then a few drops of a thin
solution of collodion is placed in the neck so as to hermetically seal
the sac. The long and contaminated ends of the cord are now cut
off, the sac dropped back into the test-tube, and the cotton stopper
replaced. (Fig. 34, 3.) The sac is now ready to be placed in the
body cavity of an animal. (Frost.)
The method of inoculation is especially 'useful in increasing the
virulence of attenuated forms, and in producing immunity in ani-
mals to induce the agglutinating and lysogenic properties in the
blood.
REFERENCES. Harris, C. f . B. I., 1902, 32 : 74 ; Frost, Proc. Am.
Pub. H. A., 1903, 28, p. 536.
Intravenous A rabbit is generally chosen for this purpose and
the inoculation made into an ear vein. Of the three branches of
the vena auricularis posterior, the ramus lateralis posterior is the
smallest, but, due to the fact that it is the most firmly imbedded in
connective tissue, it is much more easily entered than the others.
The artery forceps (Fig.
35, a) are used to gorge the
vessel and are, of course,
removed before the mate-
rial is injected. Avoid the
introduction of air, which
causes immediate death,
and keep the animals un-
der close observation for
FIG. 35. Dorsal view ot right ear of rabbit.
One hour. a, artery forceps; b, syringe.
300
MEDICAL BACTERIOLOGY
Inoculation into Lymphatic system. Fluid cultures, or suspen-
sions of bacteria, can be injected into the lymphatics by way of the
testicles, by plunging the point of the needle into the substance of
the testicle and injecting the desired amount of fluid.
Inoculation into the Pleural Cavity. Where necessary the needle
is introduced into the pleural cavity between the ribs. It is very
difficult to perform this experiment without injuring the lung.
Inoculation into the Anterior Chamber of the eye. Rarely prac-
ticed. The eye is treated with a few drops of cocaine (2% solution)
and then the needle is inserted through the cornea just in front of
its junction with the sclerotic, the needle passing into the anterior
chamber in a plane parallel to the plane of the iris.
STERILIZATION OF INSTRUMENTS. These are best sterilized by
boiling in a solution of soda or borax for 15 minutes. This is ac-
complished in an especially designed apparatus or in an ordinary
enamel stew pan. In case of emergencies the instruments may be
dipped in benzine or alcohol and burned. This is less injurious to
the instruments than heating in the direct flame.
Use blank, p. 308 for preservation of data.
OBSERVATION OF INOCULATED ANIMALS. After inoculation the
animals should be placed in separate cages, or, if placed together,
they must be described or
marked so as to be easily identi-
fied. Fig. 36 shows a simple
cage made of galvanized iron
with soldered seams. After use
it is sterilized by boiling water
in it. The wire door is covered
with a cloth to prevent the too
rapid escape of steam.
The inoculated animals must
be kept under constant observa-
PIG. 36. Wesbrook's sterilizable, gal-
vanized-iron animal cage.
tion and the following conditions noted :
a. Temperature.
~b. Loss of weight.
c. Peculiar position in cage.
d. Loss of appetite.
e. Condition of the coat or hair.
/. Condition of the secretions of the air passages, conjunctiva
and kidneys ; diarrhea or hemorrhage from the bowels.
g. The condition of the seat of inoculation.
302 MEDICAL BACTERIOLOGY
The animals should be fed regularly, weighed at the same hour
each day and the temperature taken at the rectum.
POST MORTEM EXAMINATION.
Perform -the autopsy as soon as possible after death. When
delay cannot be avoided, place the animal in the ice-chest until such
time as is convenient.
A.
a. Inspect externally and note presence and .character of any
lesion.
Z>. Sterilize a suitable post-mortem board with corrosive sub-
limate solution, 1 to 1000, place the animal belly upwards and
tack the four legs fast to the board.
c. Wash the surface of the thorax and abdomen with corro-
sive sublimate solution, make an incision through the skin at
the pubis, introducing one blade of the scissors, and extend the
incision as far as the chin.
d. Carefully dissect the skin away from the abdomen, thorax,
axillary, inguinal, and cervical regions, and fore and hind legs,
and pin it to the board as far as possible from the thorax and
abdomen. It is from the skin that the chances of contamination
are greatest.
B. All incisions from now on are made with sterilized instruments.
a. Take an ordinary potato-knife, heat it quite hot, and place
it on the abdomen in the region of the linea alba until the fascia
begins to burn ; the knife is then held transversely to this line
over the center of the abdomen, making two sterilized tracks
through which the abdomen may be opened by crucial incisions •
two burned lines are also made along the sides of the thorax.
~b. Make a central longitudinal incision from the sternum to
the genitalia with sterile scissors, the abdominal wall being held
up with sterilized forceps, or a hook, to prevent the viscera being
injured. A transverse incision is made in a similar manner.
Cut through the ribs with strong sterilized scissors along the
sterilized tracks on the sides of the thorax, when the whole ante-
rior wall of the thorax is easily lifted and entirely removed by
severing the diaphragm connections.
. c. When the thoracic and abdominal cavities are fully exposed,
a careful examination of the organs and surroundings is made
without disturbing them.
Plates (Petri-dishes) or roll cultures are prepared from the
blood, liver, spleen, kidneys, and from any exudates present.
304 MEDICAL BACTERIOLOGY
The method is as follows :
( 1 ) Heat a scalpel and scorch a small surface of the organ from
which the cultures are to be made.
(2) Heat the scalpel again and penetrate the capsule of the
organ with the point, and through the opening insert a stout ster-
ilized platinum loop, push it into the tissues, twist around, and
obtain enough material from the center of the organ to make the
culture. Cultures from blood are usually made from one of the
heart cavities, the surface being seared with a hot knife before
opening.
As soon as the culture material is obtained, cover-glass speci-
mens are prepared from each organ and from existing exudates.
Small pieces of each organ are also preserved for future exam-
ination.
When the autopsy is finished the remainder of the animal should
be burned1 and the instruments should be sterilized (see p. 300).
Wash the post-mortem board with sublimate solution. The cover-
glasses and other material likely to contain infectious matter must
also be sterilized when of no further use.
Cultures are to be incubated at 38° C., growth examined micro-
scopically, and by means of sub-cultures.
Use blank on p. 308 for preservation of data. Fig. 37 shows the
method of making a post-mortem and the location of the most im-
portant lymphatic glands.
REFERENCES. The above is taken largely from Bowhill, 74 ; see
also A. 230 ; N. 260 ; and other texts.
COMMON LABORATORY EXPERIMENTS.
The following inoculations are those most frequently made :
Streptococcus erysipelatos. Mice or rabbits, intravenous.
M. pyogenes var. aureus. Rabbit, intravenous.
Sarcina tetragena. Guinea pigs and white mice, subcutaneous.
Bacterium anthracis. Guinea pigs or rabbits, subcutaneous.
Bacterium cholerae. Rabbits and pigeons, subcutaneous.
Bacterium pneumoniae. Rabbits and mice, subcutaneous.
Bacterium pneumoniae. Rabbits and mice, subcutaneous with
sputum.
Bacterium pneumonicum. Mice and young rats, intraperitoneal.
Bacterium tuberculosis. Guinea pigs, rabbits and field mice,
subcutaneous or intraperitoneal.
1 For small animals a muffle furnace does very well.
306 MEDICAL BACTERIOLOGY
Bacterium mallei. Male guinea pigs, intraperitoneal.
Bacterium diphtheriae. Guinea pigs, rabbits and fowl, subcuta-
neous and intratracheal.
Bacillus pestis. Rats, mice, guinea pigs and rabbits, subcuta-
neous.
Bacillus Salmonii. Rabbits and mice, subcutaneous.
Bacillus tetani. Guinea pig, subcutaneous.
Bacillus tetani. White rat with garden earth.
Bacillus Welchii. Rabbit, intravenously, and then kill in 3
minutes. See p. 352.
Bacillus Welchii. Guinea pig, subcutaneous.
Microspira Metschnikovi. Pigeons, subcutaneous.
MEDICAL BACTERIOLOGY
307
MEDIASTINAL
AND BRONCHIAL"
GLANDS
LUNGS -^
LEFT LAID """"
ON RIGHT
BLADDER *'
SUBMAXILLARY
GLANDS
DEEP CERVICAL
GLANDS
AXILLARY AND
SUBSCAPULAR
GLANDS
LIVER
--ADRENAL GLANDS
— — SPLEEN
RETRO PERITONEAL
GLANDS
SUPERFICIAL
INGUINAL
GLANDS
POPLITEAL GLANDS
"-HIND GUT
FIG. 37. Diagram showing method of making autopsy on guinea pig; ana also the
Qost important glands (adapted from Delepine & Curtis).
308 MEDICAL BACTERIOLOGY
BLANK FOR ANIMAL EXPERIMENTS
Animal No Sex Age Weight
Date o'clock . , M.
Inoculated with
How inoculated
Symptoms produced :
Died (or killed) o'clock M.
Autopsy made o'clock M.
Autopsy findings :
MEDICAL BACTERIOLOGY 309
Bacteriological examination :
Histological Examination :
Organs preserved
Museum No.. Slide Nos.
310
MEDICAL BACTERIOLOGY
EXERCISE 98. PREPARATION OF TISSUE FOR EXAMINATION.
Portions of the diseased tissue, removed at autopsy, should be
cut into cubes having edges about 5 mm. long and treated as follows :
1). FIXING. Use 15 or 20 times their volume of 95% alcohol
for 24 hours. The specimens should be placed on cotton to keep
them near the top and the alcohol changed after 3 or 4 hours. If
they are not to be sectioned immediately carry to 80% alcohol.
Where larger sections are desired they should be left a longer
time in the alcohol.
2). PREPARATION FOR SECTIONING.
A.
Paraffin Method.
I
a. Absolute alcohol 6-24 hours.
I
6. Xylene 6-24 hours.
I
c. Paraffin melting at 50° C.
and kept in an oven or water-bath
at a temperature a few degrees
above the melting point of the
paraffin 3-12 hours.
I
d. Embed. Pour melted par-
affin into a paper box or other
suitable receptacle and with warm
forceps arrange block of tissue in
proper position and cool rapidly
by plunging into cold water.
I
B.
Celloidin Method
a. Mixture of ether and
absolute alcohol (equal
parts) 24 hours.
I
6. Thin celloidin (about
6%) 24 hours to several
weeks.
I
c. Thick celloidin (about
12%) 24 hours to several
weeks.
d. Remove block of tis-
sue to a piece of wood fiber
covered with thick cel-
loidin. orient, dry a few
minutes in air, then place
in 80% alcohol for 6-24
hours.
C.
Freezing Method.
I
a. Place in 1%
formalin 2 hours.
I
7). Place tissue on
plate of freezing mi-
crotome in water, or,
better, first soak tis-
sue in a syrupy so-
lution of gum arable
and moisten plate
with same before
freezing.
I
3). SECTIONING. Cut sections from 10-12 ^ thick.
4). MANIPULATION OF SECTIONS.
a. Celloidin sections can be preserved in 80% alcohol and are best
stained by placing the sections first in water and then in the stain.
The various reagents are best used in watch glasses and the sections
transferred from one to the other by means of a section lifter.
5. Paraffin sections should be fixed to the slide or cover-glass as
follows: A water-bath is heated up to a few degrees below the
melting point of the paraffin, the sections are placed on the water
where they will straighten out and are then transferred to the slide,
or, more conveniently to the cover-glass, by simply dipping the same
into the water and drawing up the section by means of the fine
point of 'a pair of forceps, or a needle, draining off the water and
drying the section in an incubator for a few hours. The sections are
more secure if the cover-glasses are first smeared with a thin coat of
312 MEDICAL BACTERIOLOGY
egg albumin. When the sections are once fixed to the cover the
staining can be carried on in the forceps as with ordinary cover-
glass preparations. Before staining, however, the paraffin must be
removed; this is done with xylene, and this in turn with absolute
alcohol.
REFERENCES. A. 182 ; M. & W. 204-239 ; N. 531.
EXERCISE 99. STAINING SECTIONS.
GENERAL HISTOLOGICAL METHOD.
Hematoxylin and Eosin.
a. Transfer sections from alcohol to distilled water.
&. Stain in alum-hematoxylin 5 minutes. The stain may be
prepared as follows (Boehmer) :
1. Hematoxylin crystals, - - 1 gram.
Absolute alcohol, 10 cc.
2. Alum, ----- 20 grams.
Distilled water, - 200 cc.
Cover the solutions and allow them to stand over night. The
next day mix them and allow the mixture to stand for one week in a
wide-mouthed bottle lightly plugged with cotton. Then filter into a
bottle provided with a good cork. The solution is now ready for
use, but its staining powers improve with age.
c. Acid alcohol 5 to 10 seconds.
d. Ammonia water (l1/^) until sections are a light blue.
e. "Wash in water.
/. Counter-stain with eosin (-^ to y2% in 60% alcohol) 3 min-
utes.
g. Alcohol, 95%, two or three changes to dehydrate and remove
excess of counter-stain.
h. Clear in oil of origanum, or in Dunham's mixture (white oil
of thyme 4 parts, oil of cloves 1 part).
i. Balsam.
GENERAL BACTERIOLOGICAL METHODS.
A. Loeffler's Universal Method.
a. Take sections out of alcohol and place in Loeffler's methylen
blue for 5 to 30 minutes.
I. Decolorize in acetic acid (0. 1%) 10 to 20 seconds.
c. Dehydrate in absolute alcohol, two or three changes, a few
seconds.
d. Clear in xylene.
314 MEDICAL BACTERIOLOGY
e. Mount in balsam.
B. Weigert' s Method.
a. Lithium carmine (carmine 3 gms., saturated aqueous solution
of carbonate of lithium, 100 cc., a crystal of thymol, filt-
ered), 5 minutes.
Z>. Acid alcohol, 15 seconds.
c. Wash in water.
d. Transfer to slide and blot.
e. Ehrlich 's anilin water gentian violet 3 minutes.
/. Blot.
g. Place in potassium iodide and iodine solution (iodine 1 part,
potassium iodide 2 parts, water 100 parts) 2 minutes.
h. Blot.
i. Decolorize in a mixture of anilin oil 2 parts and xylene 1 part,
2 to 5 minutes.
j. Blot.
Jc. Mount in balsam.
This stain can only be used with those organisms which take the
Gram stain, namely : Str. erysipelatos, M. pyogenes var. albus, M.
pyogenes var. aureus, Sar. tetragena, Bact anthracis, Bact. pneu-
moniae, Bact. rhusiopathiae, Bact' tuberculosis, Bact. leprae, Bact.
diphtheriae, Ps. aeruginosa, Bact. Welchii, B. Feseri, B. oedematis,
B. tetani and Streptothrix bovis.
SPECIAL BACTERIOLOGICAL METHODS.
Particular organisms may be stained as follows :
Pus micrococci. Loeffler 's or Weigert 's method.
Micrococcus gonorrhoeae. Loeffler 's method gives the best re-
sults.
Sarcina tetragena. Loeffler 's or Weigert 's method.
Bacterium anthracis. Loeffler 's or Weigert 's method.
Bacterium pneumoniae (Pneumococcus). Weigert 's method.
Bacterium pneumonicum (Friedlander's bacillus). The follow-
ing method is recommended for staining the capsules in sections
(M. & W.):
a. Stain for 24 hours in the incubator in the following solution :
Saturated alcoholic solution of gentian violet - 50 cc.
Distilled water ------------ 100 cc.
Glacial acetic acid - ........ 10 cc.
6. Wash out in \% solution of acetic acid.
c. Alcohol.
316 MEDICAL BACTERIOLOGY
d. Xylene.
e. Canada balsam.
Bacterium cholerae (chicken cholera). Loeffler's method.
Bacterium tuberculosis.
a. Weigert's method (staining with anilin oil gentian violet 24
hours at room temperature, or 2 to 3 hours at 40° C.).
~b. Ziehl-Neelsen 's Method.
1. Stain with carbol-fuchsin (12 to 24 hrs. room temper-
ature, 1 to 3 hrs. 40° C.).
2. Decolorize with nitric acid (10%) a few seconds, and
then with alcohol (60 to 90%) until color is nearly all extracted.
3. Counter-stain with methylen blue.
4. Dehydrate with absolute alcohol (a few seconds).
5. Clear with clove oil.
6. Xylene (and examine).
7. Mount in balsam.
Bacterium leprae.
This organism is stained with the tubercle stain, unless the sections
have been kept in alcohol for some time, in which case Weigert 's
method can be employed. To differentiate this organism from
B. tuberculosis, stain as follows :
a. An aqueous solution of fuchsin 6 to 7 minutes,
fc. Acid alcohol (nitric acid 1, alcohol 10) 14 minute.
c. Wash in water.
d. Counter-stain in a saturated aqueous solution of methylen
blue.
e. Alcohol.
/. Xylene.
g. Balsam.
The bacteria of leprosy stain readily by this method, tuber-
cle bacteria do not.
Bacterium mallei.
Slow Method.
a. Stain in Loeffler 's methylen blue 6 to 8 hours.
&. Wash in distilled water.
c. Tannic acid solution (10%) 4 to 5 hours.
d. Wash thoroughly in water.
e. Dehydrate in absolute alcohol.
/. Clear in xylene and mount.
Quick method.
a. Stain in carbol-methylen blue 10 to 30 seconds.
318 MEDICAL BACTERIOLOGY
6. Wash in distilled water.
c. Tannic acid solution (10%) y2 to 1 minute.
d. Counter-stain with a weak solution of eosin until sections are
red.
e. Wash in water until pink.
/. Dehydrate in absolute alcohol.
g. Clear in xylene and mount.
Bacterium diphtheriae. Loeffler's or better Weigert's method.
Bacillus typhosus.
a. Loeffler's methylen blue or carbol-fuchsin 15 min. to 24 hrs.
b. Wash slightly in distilled water.
c. Place in 30% solution of tannic acid for 10 to 60 min.
d. Dehydrate rapidly in alcohol.
e. Clear in xylene.
/. Examine.
g. Mount in balsam.
Such sections examined under a low power will be found to con-
tain heavily stained masses, which under a high power prove to be
clumps of bacilli. Not infrequently the bacilli are difficult to detect
in tissue from typhoid cadavers.
Bacillus Salmonii (hog cholera). Loeffler's method.
Bacterium WelcJiii ( gas bacillus) . Weigert 's and Loeffler 's methods.
Bacillus Feseri (symptomatic anthrax). Use Pfeiffer's stain:
a. Dilute carbol-fuchsin % hour.
&. Absolute alcohol slightly acidulated with acetic acid until
section is a reddish violet tint.
c. Xylene and examine.
d. Mount in balsam.
Bacillus oedematis (malignant oedema). Pfeiffer's stain.
Streptothrix bovis (actinomyces).
a. Ziehl's carbol-fuchsin, 10 minutes.
6. Wash in distilled water.
c. Picric acid (cons. ale. solution).
d. Wcsh in distilled water.
e. Wash in alcohol (50%).
/. Dehydrate in absolute alcohol.
g. Clear in xylene.
h. Balsam.
Tissue stained yellow, rays red.
REFERENCES. M. & W. 239-286 ; N. 537.
320
MEDICAL BACTERIOLOGY
CHAPTER IX
BACTERIOLOGICAL DIAGNOSIS
EXERCISE 100. EXAMINATION OF BUCCAL SECRETION.
DEFINITION. The secretion of the mouth, or saliva, is a mixed
product derived in part from the mucous glands within the mouth,
and also from the parotid, submaxillary and sublingual glands. In
disease the normal character of the different parts may varjr, or
there may be various exudates and growths present.
COLLECTION. Material for bacteriological examination is best
obtained by means of a sterile probang or by forceps. This material
may be examined directly by means of cover-glass preparations or
by means of cultures.
a. Method of Preparing Outfit. Wind a small piece of absorbent
cotton on the end of a wire (about 1 mm. in diameter and 14 cm.
long). Thrust the other end of the wire through
the cotton plug of a test-tube or fasten in a cork and
sterilize at 150° C. for 1 hour. This with a tube of
nutrient medium (usually Loeffler's Blood serum) is
placed in a box for transportation. Fig. 38.
&. Method of Using Outfit, The patient is placed
in a good light and the probang gently but firmly
rubbed over the suspected area of the throat and then
drawn gently over the surface of the medium, both Jj
tubes securely stoppered and the outfit sent to the
laboratory.
Tube 1 is a ster-
_ _. _, ile swab; 2 is a
ORGANISMS COMMONLY FOUND. blood serum
slope.
Bacterium diphtheriae.
The presence of this germ in the mouth usually results in the
formation of a pseudo-membrane, a portion of which is to be removed
with a pair of forceps, or by means of the outfit described above. It
should be examined directly for the diphtheria bacillus by smearing
on a cover-glass and staining by following methods :
322 MEDICAL BACTERIOLOGY
a. Loeffler 's methylen blue, or Roux stain.1
&. Gram's stain.
c. Neisser's stain: a. 1 gram methylen blue dissolved in 20 cc.
of alcohol (96%), is added to 950 cc. of distilled water and 50 cc.
of glacial acetic acid; &. 2 grams of bismarck brown dissolved in a
Ijter of distilled water. Films are stained in a. 5 to 8 seconds, washed
in water, stained in Z>. 3 to 5 seconds, dried and mounted. The
Crouch2 stain may be similarly employed.
Usually, however, mere microscopical examination is not suffi-
cient, and culture methods must be employed. In fact this method
ought always to be used. In this case make smears on Loeffler 's
blood serum and incubate them at 36 to 38° C. for 12 to 24 hours and
then examine the growth in cover-glass preparations. The diph-
theria organism if present, should show :
a. Characteristic appearance with Loeffler 's methylen blue.
ft. Positive Neisser stain.
c. Positive Gram stain.
Occasionally micro-organisms (pseudo-diphtheria bacilli among
others) are met with that very closely resemble the Klebs-Loeffler
bacillus and render a positive diagnosis doubtful. In such cases
attention to following table will be helpful :
xBoux stain— Solution A: Dahlia 1, alcohol 10, and distilled water 90
parts; Solution B: Methyl green 1, alcohol 10, and distilled water 90 parts;
mix 2 parts of A with 1 of B.
8 Crouch stain — Aqueous solution of dahlia (1%) 1 part, aqueous solution
of methyl green (1%) 5 parts, and distilled water 4 parts.
324
MEDICAL BACTERIOLOGY
B. diphtheriae
B. pseudo diphtheriticum
1) Form
Slender, and of same di-
Thicker at center than
ameter throughout
ends, plumper, shorter
and less variable than B.
diphtherias
2) Size
Average 1.2 to 2 p
Averaging 1 to 1.6 /x
3) Threads
Not formed
Not formed
4) Grouping
Parallel grouping more or
Parallel but lie closer to-
less characteristic but do
gether
not touch
5) Involution forms
Common
Rare
6) Motility
Immotile
Immotile
7) Stains
a. Loeffler's methylen
Stains readily giving band-
Stains more regularly
blue
ed or polar stain
Polar stain rare
6. Gram.
Positive
Positive
c. Neisser
Characteristic stain with
Not under 24 hours
very young cultures, six
hours
8) Spores
Absent
Absent
9) Alkaline potato
Growth almost invisible
Visible and cream colored
10) Sugar agar and gela-
in 2 days
tin stab cultures
Full length of stab
Only at upper part
11) Neutral litmus milk
Acid reaction
Alkaline reaction
12) Dextrose bouillon
Acid reaction
Alkaline reaction
13) Anaerobic cultures in
H.
Grows well
No growth
14) Nitroso-indol reaction
After 7 days
After 21 days
15) Inoculation experi-
ments (Guinea pig
subcutaneous)
Death 36-48 hours
Non-pathogenic
Pus Micrococci. (Str. erysipelatos, M. pyogenes var. aureus and
albus, Sar. tetragena.)
a. Stained cover-glass preparations are to be examined, and if
micrococci are found make :
6. Smear cultures, or better, agar plate cultures and work up the
colonies as they appear.
Monilia Candida (Organism of Trush).
The material is collected by removing a portion of the patches or
membrane and examining it :
a. Under the microscope in a drop of glycerine.
~b. Cover-glass preparations stained with carbol-fuchsin or Gram's
method.
c. By means of smear cultures on agar or blood serum, the result-
ing growth being examined either in glycerine mounts, or stained
cover-glass preparations.
KEFERENCES. Em. 43 ; v. J. 95 ; Si. 122. See also various texts
under special organism.
326 MEDICAL BACTERIOLOGY
EXERCISE 101.— EXAMINATION OF SPUTUM.
DEFINITION. By this term is meant all of the material derived
from the air passages by the act of coughing or hawking.
METHOD OF COLLECTION. For diagnostic purposes it is best col-
lected in a salt-mouthed bottle (about 2 oz. capacity) which has been
sterilized. The morning sputum is best, and, before being collected,
the mouth should be rinsed out with water.
ORGANISMS MOST COMMONLY FOUND.
Bacterium tuberculosis. Place the sputum in a Petri dish over a
black surface and select one of the little cheesy masses, if these be
present, and smear it on a cover-glass. Where these particles are
not present a loop or two of the thick portion is used. The cover-
glass preparations are to be stained by one of the following methods :
a. Gabbett, see Part 1, p. 62.
&. Ziehl-Neelsen :
1. Carbol-fuchsin ten times through the flame (5 to 10 min.).
2. Nitric acid (30%) momentarily.1
3. Water.
4. Alcohol (60%) until red color disappears. It may be
necessary to immerse preparation in acid a second time, but care
must be exercised to prevent extraction of dye from tubercle bac-
terium.
5. Loeffler's methylen blue, 1 minute.
6. Mount and examine.
While the tubercle bacteria may be detected when present in
considerable numbers with a 1-6-inch objective, when there are few
present, a ^-inch oil immersion will be necessary, and this ought to
be used to search all slides where the tubercle germ has not been
found with a lower power. A mechanical stage is a great conven-
ience in a systematic search.
At least two preparations should be stained and thoroughly exam-
ined before a negative result is pronounced.
The viscosity of sputa may be overcome and the bacteria con-
centrated, where the number is very small, by 1) Ribbert's method
which consists in the addition of a 2% solution of caustic potash and
boiling. This dissolves the mucus, and the bacteria are then de-
posited with the sediment. This sediment can be obtained by allow-
1 Eavenel recommends use of 5% nitric acid in 80% alcohol, claiming that
there is no danger of decolorizing the tubercle bacilllus no matter how long
the contact.
328 MEDICAL BACTERIOLOGY
ing the mixture to stand in a conical glass vessel or, more quickly, by
the use of a centrifuge. 2 ) Hammond 's method :
a. Add 5% of crystallized carbolic acid (in the case of sputum
add 5 times its bulk of a 5% solution of carbolic acid).
&. Place 15 cc. in the tubes of a centrifuge and whirl for 15
minutes.
c. Pour off supernatant fluid and treat precipitate with 3 cc. of a
6% KOH solution. Mix thoroughly and allow to stand 2 minutes.
d. Fill to 15 cc. mark with distilled water and whirl 20 minutes.
e. Make cover-glass preparation of sediment (or purify same by
repeated washings and centrif legalizations with distilled water).
A centrifugal machine should be able to make at least 2,500
revolutions per minute. This speed ought to be maintained for 15
minutes. Sputum may be preserved by addition of a small quantity
of carbolic acid (5%).
Negative results are of positive diagnostic value only when re-
peated examinations are made of different samples taken at different
times.
EEFERENCES. Em. 75.
Bacterium influenzae. This micro-organism is frequently pres-
ent in enormous numbers (100 or more) and sometimes in almost
pure cultures in the greenish purulent masses in the sputum/ It
stains readily with the ordinary dyes, and when lightly stained
presents the bipolar stain. Carbol-fuchsin diluted 10 times with
distilled water is one of the best stains. Gram 's stain is negative.
Sputum from suspected cases should be collected either by means
of a probang, or in a bottle, and examined :
1) Microscopically by staining, with a weak carbol-fuchsin,
smears from the purulent masses. If a very small bacillus is in
large clumps, which fails to retain stain by Gram's method, the
evidence is strong that it is the influenza bacillus; the diagnosis
should be confirmed, however, by
2) Cultures on blood agar.
Animal inoculations are without effect.
REFERENCES. Em. 64.
Bacterium pneumoniae (pneumococcus).
The sputum of patients suffering from pneumonia is usually of
330 MEDICAL BACTERIOLOGY
a rusty color, due to presence of blood ( rusty sputum ) . The ' ' pneu-
mococcus ' ' is readily seen in such material when stained by Gram 's
method or with carbol-fuchsin and momentarily washed with alco-
hol, as lancet-shaped organisms with outer ends pointed and sur-
rounded by a clear area— the capsule. The capsule can be easily
stained by Welch 's method. ( See 27. )
This organism is also frequently found in the sputum of healthy
persons and small numbers may be detected by means of animal
inoculation. The rabbit or mouse is most susceptible and should be
inoculated subcutaneously. As a result of infection with this organ-
ism the animal dies quickly with a typical septicemia, the micro-
organisms being found in great numbers in the blood current.
Bacillus pestis. This micro-organism is frequently found in the
sputum, especially in the pneumonic form of the disease— for meth-
ods of detection see 105.
Streptothrix bovis (actinomyces). This organism has been occa-
sionally found in sputum and in such cases the peculiar morphology
of the colonies is well brought out by Gram 's method. See 105.
REFERENCES, v. J. 114; Si. 245. See also various texts under
particular organisms.
EXERCISE 102. EXAMINATION OF BLOOD.
COLLECTION. For serum test (Widal reaction) the blood may be
collected and dried (see below), but in other cases where cultures are
to be made, the blood must be collected aseptically in sterile recepta-
cles and hermetically sealed. For this purpose Sternberg's bulb is
excellent. The skin should first be sterilized by use of corrosive sub-
limate or carbolic acid followed with alcohol.
It is usually well in any case to make cover-glass smears at the
bed-side for microscopical examination. These are best made as
follows: Place a drop of blood about the size of a pin head on a
perfectly clean cover-glass and then place a second cover-glass on
this ; this flattens the drop of blood out into a thin film. Immedi-
ately and before coagulation can take place the two are drawn apart
horizontally and the films allowed to dry. ( Cabot. )
Bacterium anthracis. In case of animals dead of suspected an-
thrax, blood or portion of spleen should be removed with least pos-
332 MEDICAL BACTERIOLOGY
sible danger from infection or distribution of bacilli and studied as
follows :
a. Microscopical examinations of blood or of the spleen pulp of
animals show (when stained with Loeffler's methylen blue) large
bacteria in chains (5 or 6 segments) presenting the bamboo appear-
ance.
~b. In hanging drop preparation large, homogeneous, immotile
bacilli.
c. Agar plate cultures should also be made, and, from the separate
colonies, subcultures ; the gelatin stab being especially characteristic.
d. In important cases (as in man) guinea pigs, or white mice,
should be inoculated, and, in case of death, organism isolated and
identified.
Spirockaeta Obermeieri (relapsing fever). This organism is
found in the blood only during a paroxysm. It is a long slender
organism 6 or 7 times the diameter of a red blood corpuscle (45/*).
It has a brisk, vibratile movement in the direction of its long axis,
and is very sensitive to reagents of all kinds. Even the addition of
distilled water will cause it to disappear. Fresh blood is best, but
dried smears may be used and stained with f uchsin, or by Gunther 's
method :
a. Dried films are treated with acetic acid (5%) 10 seconds, this
is removed by blowing and holding film over flask of strong ammonia
previously shaken.
&. Stained in Ehrlich's gentian violet.
c. Washed with water.
d. Dried.
e. Mounted in balsam or xylene.
/. Examined.
Pus Micrococci. These are occasionally found, and for method
of detection see 105.
Bacterium mallei. Sometimes found in the blood of those suffer-
ing with glanders. It may be detected in the blood- smears. For
special methods see 105.
Bacterium pneumoniae (pneumococcus) . This germ is frequent-
ly present in fatal cases 24 to 48 hours before death. The blood
should be drawn with a sterile hypodermic syringe and about 1 cc. of
blood mixed with a tube of melted agar at 43° C. and poured into a
Petri dish. Characteristic colonies appear in 24 to 48 hours.
334 MEDICAL BACTERIOLOGY
Bacterium tuberculosis. In case of miliary tuberculosis they
may be very rarely found in sufficient numbers to be detected by
staining methods, see sputum 101.
Bacterium influenzae. Canon claims to have stained and culti-
vated this organ in blood, but this needs confirmation.
Bacillus coli. This organism may be found in the blood. For
methods of isolation and identification see f eces 103.
Bacillus pestis. This germ occurs in the blood, in certain cases
at least. Considerable skill in detecting it is required— due to its
variable appearance. Broth tubes should be infected and animals
inoculated.
Bacillus Salmonii (hog cholera).
a. Make agar plate and streak cultures from spleen of dead ani-
mal, and work up the colonies as they appear.
b. Widal Reaction (for technique see below under B. typhosus).
Plasmodium malariae.
a. Examination of fresh blood. A droplet of blood from finger,
or from lobe of ear, is placed on a glass slide, covered with
a cover-glass and then the cover-glass is ringed with vaselin.
Examination should be made with a -fa in. oil immersion.
b. Stained. Prepare films as directed above and stain with
methylen blue and eosin, or treat films with a very weak
acetic acid, 2 or 3 drops to 30 cc. of water ; to remove hemoglo-
bin, wash with water and stain with following solution for y2
minute :
Borax 5.0 parts.
Methylen blue 0.5 parts.
Water 100 parts.
Wash, dry and mount in balsam (Manson).
REFERENCES, v. J. 45 ; Si. 100. See also texts under particular
organisms.
WIDAL REACTION. Dried blood method. This method is espe-
cially valuable where patient is some distance from the laboratory.
Collect the blood as follows: "Wash with boiled water the part
from which the blood is to be obtained (lobe of ear, end of finger, or
toe in infant). Prick deeply the skin with a needle, " Remove two
or three large drops of blood on a clean glass slide, aluminum foil,
piece of isinglass or letter paper. Allow the blood to dry. Then
place in an envelope and send to laboratory and test as follows :
$36 MEDICAL BACTERIOLOGY
a. Make a hanging drop preparation from a 24 to 72-hour old
agar, or bouillon, culture of Bacillus typhosus.
5. If the bacilli be actively motile, remove the cover-glass, add
to the culture a small drop of a solution of typhoid blood (diluted
from 10-50 times), return the cover glass to the slide and seal well
with vaselin.
c. Examine with a high dry power (1-6 in obj.) rather than with
the oil immersion.
The dilution is made in the following way : Nine drops of sterile
water are placed around the drop of dried blood. (The drops of
water should be of about the same size as that of the original drop of
blood.) The drops are all mixed together and allowed to soak up
the blood for about two minutes. In this way an approximate dilu-
tion of one to ten is obtained. One drop of this is added to the
hanging-drop culture. This gives a dilution of one to twenty which
is the one usually employed.
More exact dilutions of dried blood may be made by weighing out
the blood and adding it to a measured amount of water.
Where possible the blood should be collected so that the clear
serum may be separated and used for the test. This can be done in
hospital work and wherever it is possible to get the blood to the
laboratory a few hours after it is collected. For this purpose a glass
pipette is prepared by drawing out a glass tube, as indicated
in Fig. 39, which represents the pipette natural size. The
skin is cleaned and the blood drawn as indicated above and
when a large drop has collected on the skin one of the points
of the pipette is introduced when the blood is drawn up by
capillary attraction. The bulb ought to be about one-half
filled. The pipette is then placed in a horizontal position
until the blood has clotted, when it may be taken to the
laboratory. It should then be placed in the ice chest, still
in a horizontal position, for two or three hours. The end
which was used to draw up the blood is then scratched with
a file and broken off. By holding the tube in a vertical
F position the clear serum may now be dropped from the
B. ! oo a opposite end into a glass or porcelain capsule. The clear
plp6trtJG«
serum is then taken up with a clean capillary pipette and a
drop placed in another capsule and then after rinsing out the same
pipette is used to add the requisite number of drops of bouillon or
salt solution to make the required dilution. The test is then made
in exactly the same way as described for the dried blood,
338 MEDICAL BACTERIOLOGY
In a typical reaction the motility is almost immediately affected,
and soon motion ceases altogether while the bacilli collect in clumps,
FIG. 40. Widal Reaction. I. B, typhosus before adding typhoid blood; II, A typical
reaction.
i. e., become "agglutinated." (Fig. 40.) The usual time limit is
thirty minutes when the dilution is 1 to 50.
REFERENCES, v. J. 45 ; Si. 100. See also texts under particular
organism.
EXERCISE 103. EXAMINATION OF FECES.
The material expelled from the rectum and comprising the sub-
stances from the food and the secretions of the alimentary tract come
under this head. The number of micro-organisms occurring here is
enormous, and comprise a large number of species and among them
several pathogenic forms particularly B. typhosus, Msp. comma,
Bact. tuberculosis and Amoeba coli.
Bacillus typhosus. This organism occurs in the f eces in the case
of typhoid patients; but on account of the large number of other
organisms its detection is very difficult. The following methods are
the most serviceable :
A. PARIETTI'S METHOD. This method consists in adding Parietti's
solution (carbolic acid 5 grams, hydrochloric acid 4 grams, and dis-
tilled water 100 cc. ) to bouillon in the following manner : A num-
ber of tubes of bouillon have a varying quantity of the above solu-
tion added, e. g. 1 drop to one tube, 2 to another, 3 to another, and
so on. These tubes are inoculated with a small quantity (one or two
loops), of the feces and then placed in the 38° C. incubator. Twen-
340 MEDICAL BACTERIOLOGY
ty-four hours later the tube containing the largest amount of Pari-
etti's solution which shows growth probably contains B. coli and B.
typhosus if it is present. The organisms may be separated most
quickly and easily by the use of the lactose litmus agar plate. The
blue colonies should be worked up, and especially tested for their
agglutinating power on typhoid blood. Instead of the lactose litmus
agar one of the following media may be used :
B. Hiss' PLATE MEDIUM.1 This contains:
10 grams of agar.
25 grams of gelatin.
5 grams of beef extract (Liebig).
5 grams of sodium chloride.
10 grams of dextrose.
1000 grams of water.
It is made by first dissolving the agar, salt and extract in the
water, then the gelatin is added and dissolved, the reaction changed
by use of NaOH and phenolphthalein so that it will contain not less
than 2% normal acid, cleared with two eggs and filtered, dextrose
added and the medium tubed and sterilized.
Make plate cultures in ordinary way and incubate at 38° C. for
18 hours, then examine the colonies microscopically. The colonies
of B. typhosus have irregular outgrowths and fringing threads. The
colonies of B. coli, on the other hand, are much larger and as a rule
are darker in color and do not form threads.
The colonies may be further examined by the use of Hiss' Tube
Medium.
5 grams of agar-agar.
80 grams of gelatin.
5 grams of beef extract (Liebig).
5 grams sodium chloride.
10 grams dextrose.
1000 grams water.
Made as plate medium except that it is to contain 1.5% normal
acid.
Within 18 hours at 38° C. the typhoid bacilli produce a uniform
clouding. The colon bacilli do not produce uniform clouding and
do produce gas.
C. MEDIUM OF MACCONKEY, as modified by Griinbaum.8
i Jour. Exp. Med. 1897, 2: 677.
* Brit. Med. Jour. 1902, Pt. 1, p. 1473.
342 MEDICAL BACTERIOLOGY
Twenty grams each of agar-agar and peptone are dissolved in
one liter of boiling water, and the whole made alkaline by adding 4
cc. of a normal solution of sodium hydrate after having first brought
the reaction to the neutral point of litmus.
Then add, while hot,
Sodium taurocholate .----.... 5 grams.
Lactose 10 grams.
Neutral Red (5% watery solution) 10 cc
When the solution is complete the mass is filtered through cotton,
tubed, and sterilized in the steam sterilizer once for twenty-five or
thirty minutes.
D. MEDIUM OF DRIGALSKI AND CONRADI.'
To two liters of -sugar-free broth add:
Peptone (Witte) - - - 10 grams
Nutrose 10 grams )
e, -,. 1,1 -j -i A > these may be omitted
Sodium chloride - - 10 grams [
and dissolve by the aid of heat. The mixture is brought to the boil
and sixty grams of agar-agar added, and the mixture kept boiling
until the agar is dissolved. Then the reaction of the mass is made
weakly alkaline to litmus by the addition of sodium hydrate (4%
sol.) and filtered.
This being done, a mixture of litmus solution (6%) and lactose
(c. p.)
Litmus sol. ------...... 260 cc.
Lactose 30 grams.
is added while both solutions are hot, and the whole boiled gently for
five minutes. Then add a solution of water-free sodium carbonate
(10%) in the proportion of 4 cc. (this may be omitted) followed by
20 cc. of a fresh solution of crystal violet (Griibler's)— 0.1 gram in
100 cc. water— tubed, and sterilized in the steam sterilizer for 20
minutes on three successive days.
All suspected cultures should be tested with typhoid blood
(Widal reaction).
The typhoid organism may be isolated from the stools during the
first two weeks of the disease.
Microspira comma (Asiatic cholera).
1. Microscopal examination of "rice-water" discharges for
spirilla lying parallel.
2. Culture methods. Gelatin or agar plates should be made
from the rice-like flakes ; other flakes should be inoculated into flasks
* Zeit. f . Hyg., 1902, Heft ii, p. 283.
344 MEDICAL BACTERIOLOGY
of peptone water (Dunham's solution) and incubated at 38° C.
The surface growth 6-12 hours later is to be examined microscopi-
cally and by means of plates. Then test the peptone cultures for
nitroso-indol (cholera red reaction) by the addition of a few drops
of sulphuric acid.
B. dysenteriae. This organism has been isolated from the feces
of dysentery patients by numerous investigators and from children
suffering from summer diarrhoea by Duval & Bassett1 and others.
The following method is recommended :
Agar plates are made from the bloody mucus in the feces or
from scrapings of the ulcerated mucosa of the intestines. Agar
plates are made and incubated at 38° C. for 12 hours and then the
colonies which have appeared are marked with a pencil or pen and
then the plate is incubated for several hours longer. The colonies
which appear later are most likely to be colonies of B. dysenteriae.
The suspected colonies are then put into dextrose agar and only
those which fail to produce gas are tested farther. The crucial test
is the Widal reaction which can be made with blood obtained from
the patient or cadaver.
Bacterium tuberculosis. This organism has been found in the
stools in cases of intestinal ulcerations, and may come, in cases of
phthisis, from ingested sputa.
Ameba coli.
a. A drop of the mucus portions of stool is placed on a glass
slide, covered with a cover-glass and examined with a magnification
of about 500 diameters (1-6 in objective). Examination should be
conducted on a warm stage in order to get ameboid movements.
Z>. Preparations may be stained with methylen blue and carmine.
The nucleus is stained with carmine.
c. Discharges may be hardened and stained by Mallory 's method
as follows :
1. Fix tissues in alcohol.
2. Stain (paraffin) sections in a saturated aqueous solution of
thionin for 5-20 minutes.
3. Wash in water.
4. Differentiate in a 2% aqueous solution of oxalic ocid %-l
minute.
5. Wash in water.
1 Duval and Bassett, Amer. Med., 1904, 4: 417.
346 MEDICAL BACTERIOLOGY
6. Dehydrate in alcohol (95%),
7. Clear in oil of bergamot.
8. Wash with xylene and mount in balsam.
Nuclei of Amebae brownish red, other nuclei blue.
REFERENCES, v. J. 199 ; Si. 206. See also texts under various
organisms.
EXERCISE 104. EXAMINATION OF URINE.
For bacterial examination urine should be drawn with a sterile
catheter into a sterile bottle.
Bacterium tuberculosis.
For method of staining see under Sputum, 101.
It is best to centrifuge the product and care must be taken to
differentiate from the smegma bacterium. For this pur-
pose stain cover-glass smears as follows (Bunge & Fran-
teroth) :
a. Absolute alcohol, 3 hours.
b. Chromic acid, 15 minutes.
c. Stain in hot carbol-fuchsin.
d. Decolorize in sulphuric acid (25%) 2-3 minutes.
e. Counter-stain with a saturated alcoholic solution of methylen
blue.
The smegma bacillus is decolorized by this method.
The tubercle bacterium in urine is frequently present in clusters
while the smegma bacterium occurs singly. Injection of guinea
pigs, smegma bacillus is non-pathogenic.
The following organisms have also been found in the urine. For
methods of isolation see references.
Pus Micrococci. 105.
Micrococcus gonorrhoeae. 105.
Bacillus typhosus. 103.
Spirochaeta Obermeieri (relapsing fever). 102.
REFERENCES, v. J. 273; Si. 500, and texts under the various
organisms.
EXERCISE 105. EXAMINATION OF TRANSUDATES AND EXUDATES.
The material should be collected in sterile vessels under aseptic
precautions. Make several cover-glass preparations and stain one
with Loeffler's methylen blue and the others with gentian violet or
carbol-fuchsin. Mount and examine.
348 MEDICAL BACTERIOLOGY
a. If staphylococci alone are present search for the Pus Coccus Group.
6. If streptococci suspect Str. erysipelatos.
c. If diplococci or tetracocci.
1. Within the pus-cells test for M. gonorrhoeae or M. Weichsel-
baumii.
2. Free suspect Sar. tetragena.
d. If bacilli any of the following may be searched for:
1. B. coli. This organism is likely to be found especially in suppu-
rative peritonitis and diseases of the urinary organs. 2. Bact. anthracis.
3. Bact. pneumoniae. 4. Bact. tuberculosis. 5. Bact. leprae. 6. Bact. mal-
lei. 7. B. pestis. 8. Ps. aeruginosa. 9. Bact. Welchii. 10. B. oedematis.
11. B. tetani.
e. Streptothrix bovis.
/. Ameba coli.
Pus Micrococci. These organisms are frequently present in .pus
and should be isolated and identified in pure cultures, as micro-
scopical examinations alone will not suffice.
Streptococcus erysipelatos. This organism is not infrequently
present and can be readily identified by culture methods.
Micrococcus gonorrhoeae. Pus should be collected in a sterile
receptacle or spread on cover-glasses and allowed to dry. When
once dried it should not be wet or moistened again as this would
destroy the pus-cells, and hence the value of the material for diag-
nosis.
a. Simple stain.
1. Loeffler's methylen blue 3-5 minutes.
2. Wash in water.
3. Dry, mount in balsam and examine with -fa in. oil immer-
sion.
4. Look for a biscuit-shaped diplococcus within the pus cells,
fe. Gram's method.
1. Anilin oil gentian violet 15 minutes.
2. Wash in water.
3. Treat with iodine solution 2 minutes.
4. Decolorize with alcohol.
5. Counter-stain with eosin, y2 minute.
6. Wash, dry and mount in balsam.
7. Examine with oil immersion.
If the gonococci be present they will be stained brown.
If diagnosis be of great importance make cultures as follows :
a. Make 6 or more streak cultures on blood agar, or better, make
plates on Wertheim's medium (p. 158). Grow at 38° C.
350 MEDICAL BACTERIOLOGY
6. Make a set of ordinary agar plates, or streak cultures, and
keep at 38° C.
The gonococcus grows on the first two media, but not on the plain
agar. The gonococeus is the only organism that :
1. Occurs in groups (cell-colonies) in pus-cells.
2. Is decolorized by Gram's method.
3. Does not grow on agar at room or blood heat.
Micrococcus Weichselbaumii (M. intracellularis).
Pus may be obtained by lumbar puncture which is performed as
follows : The back of the patient and the operator 's hands should
be made sterile. The needle (4 cm. X 1 mm. for children) should
be boiled 10 minutes. The patient should lie on the right side, with
the knees drawn up and the uppermost shoulder so depressed as to
present the spinal column to the operator. The puncture is gen-
erally made between the third and fourth lumbar vertebrae. The
thumb of the left hand is pressed between the spinous processes, and
the point of the needle is entered about 1 cm. to the right of the
median line, and on a level with the thumb nail, and directed slightly
upwards and inward, toward the median line. At a depth of 3 or
4 cm. in children and 7 or 8 in adults the needle enters the subarach-
noid space and the fluid flows usually by drops. This is allowed to
drop into an absolutely clean test-tube, which has previously been
plugged and sterilized. From 5 to 15 cc. of the fluid is a sufficient
quantity for examination. Cultures should be made at once on
blood agar and on plain agar (M. & W. 371). After standing some
hours, the sediment should be examined in cover-glass preparations,
stained with Loeffler's methylen blue and by Gram's method.
Microccus Weichselbaumii stains by Loeffler's method and ap-
pears as a diplococcus in groups in the pus cells, is decolorized by
Gram's method, and grows on blood-agar, and feebly on ordinary
agar at 38° C.
The following organisms are also found occasionally. For meth-
ods of diagnosis see exercises indicated.
Bacillus coli. 103.
Bacterium tuberculosis. 101.
Bacterium leprae. For method of staining, see 99.
Bacterium pneumoniae. Stain for capsule. Cultivate on blood-
agar. 101.
Bacterium mallei.
a. Widal reaction. (If in man, typhoid and diphtheria must be
excluded in case of a positive reaction.)
352 MEDICAL BACTERIOLOGY
b. Examination of discharge.
1. Microscopical examination usually without result.
2. Cultures, glycerine agar and potato from pus.
c. Animal inoculations, Straus' method.
Bacillus pestis.
a. Make plate cultures from blood and buboes and work up
colonies.
b. Make subcutaneous inoculation into guinea pigs from bubo,
and if death ensues search for B. pestis.
Pseudomonas aeruginosa (B. pyocyaneus). Easily recognized
by its culture characters.
Bacterium Welchii (gas bacillus).
This germ is non-pathogenic for rabbits, but Welch and Flexner
have shown that if a rabbit be inoculated intravenously with 0.5 to
1 cc. of a bouillon culture and killed after a lapse of 5 or 10 minutes.
and the animal kept at 18°-20° C. for 24 hours or at30°-35° C. for
4 to 6 hours, the organism will multiply in the blood and produce
large quantities of gas in the vessels and organs. This effect is
characteristic.
Bacillus oedematis (B. malignant oedema).
a. Make cover-glass preparations from fluid of affected parts.
b. Also make anaerobic cultures. If material contains spores It
should be heated to 80° C. for 10 minutes before it is seeded.
Bacillus tetani.
a. Make cover-glass preparations from pus and search for drum-
stick bacillus.
b. Make dextrose bouillon and agar-plate cultures and develop
in hydrogen.
c. Inoculate animals with the discharge, and also with the bouil-
lon culture, and watch for characteristic symptoms.
Streptothrix bovis (actinomyces).
a. Place one of the minute sulphur-yellow nodules in a drop of
glycerine on a glass slide and then apply gentle pressure.
b. Even the low powers of a compound microscope will then show
something of the clustered arrangement which can be more carefully
studied under a higher power.
c. Intraperitoneal inoculation of guinea pig. One month later,
nodules on peritoneum.
Ameba coli. 103.
REFERENCES, v. J. 405 ; Si. 514 and 518. See also texts under
the various organisms.
354 MEDICAL BACTERIOLOGY
EXERCISE 106. DIAGNOSIS OF RABIES.
A. Microscopical Diagnosis.
a. The head of the animal is opened and the brain removed.
In case the animal is some distance from the laboratory it is best to
cut off the head, pack in ice and ship by express.
b. Thin pieces of the various parts of the brain, such as
Ammon's horns, cerebellum, cerebrum and medulla are fixed in
equal parts of formalin and 95% alcohol for 12 to 18 hours. They
are then treated as follows:
1. 95% alcohol, y2 hour.
2. 95% alcohol again for % hour.
3. Anilin oil until clear ; one hour is usually sufficient.
4. Xylol 15 to 30 minutes.
5. Melted paraffin, 2% hours.
6. Cut to 3 or 4 microns.
7. Stain in hematoxylin and eosin.
Cell inclusions known as negri bodies, which are usually found
most abundant in Ammon's horns, are the evidence of rabies.
The medulla is searched for infiltrations around the blood ves-
sels. These changes are found in a few conditions other than
rabies.
B. Smear Method.1
a. Small pieces of the various parts of the brain are crushed out
between two clean slides and treated as follows:
1. Fix the smears while still wet in methyl alcohol (neutral-
ized with sodium carbonate) to which yV % °^ picric
acid has been added. Blot off excess of fixative.
2. Stain smears as follows:
Saturated alcoholic solution of fuchsin, 3 cc.
Saturated alcoholic solution of methylene blue, 2 cc.
Distilled water, 30 cc.
Heat the slide over a flame until it steams, wash in tap
water and blot. The stain does not keep long.
1 Williams, Amer. Jour, of Public Hygiene, Feb., 1908.
356 MEDICAL BACTERIOLOGY
The negri bodies are often found outside of the cell and can be
recognized by their form, color, and internal structure.
C. Pasteur 's Method.
a. The medulla of the suspected animal is removed under aseptic
precautions, as soon as possible after death.
b. Place a piece of the medulla about the size of a pea, in 4 or
5 cc. of sterile bouillon and thoroughly grind up the same.
c. Anesthetize a rabbit with ether, clip the hair from between
the eyes and ears and disinfect with a carbolic acid solution.
d. Make a longitudinal incision through the skin and subcutan-
eous tissue along the median line, while a crucial incision is made
through the periosteum on one side of median line thus avoiding
hemorrhage from the longitudinal sinus. The periosteum is then
pushed back and a disc of the skull (14 inch in diameter) removed
with a trephine and the dura mater exposed.
e. With a sterile hypodermic syringe introduce 2 or 3 drops of
the suspension of medulla beneath the dura mater, stitch the skin,
dry, and seal the wound with collodion.
The rabbits apparently experience no inconvenience ; the wound
heals rapidly and the rabid symptoms appear in from 15 to 30 days,
although sometimes they may occur earlier or much later.
EXERCISE 107. EXAMINATION OF MATERIAL FROM HUMAN
AUTOPSIES.
At human autopsies smears from the organs should be made on
cover-glasses and afterwards stained and examined. Plate-cultures
should also be made from the various organs. In all cases the sur-
face from which the material is to be obtained should first be burned
to avoid infection of cultures with extraneous germs. Portions of
the various organs should also be preserved and hardened in alcohol.
358 MEDICAL BACTERIOLOGY
CHAPTER X
DETECTION OF PATHOGENIC BACTERIA IN
WATER AND MILK SUPPLIES
EXERCISE 108. EXAMINATION OF WATER FOR PATHOGENIC
BACTERIA.
It is rarely necessary to test water directly for either the typhoid
or cholera organisms, as there is little chance of their being found
except in the most grossly polluted waters. What is usually sought
for is evidence of sewage pollution. If this is found the water is
not regarded as potable. The more common methods of detecting
fecal bacteria have already been given (Chapter V). The follow-
ing methods are reliable and the detection of these germs in artifi-
cially infected waters furnish most excellent practice for the student.
Bacillus typhosus. In the examination of water it is best to con-
centrate the bacteria by filtering a large amount of the water through
a Berkefeld filter and to use the slime on the filter to make the plates.
a. Parietti's method, see 103.
~b. Hiss' method. Make plate cultures and incubate at 38° C.
for 18 hours. Inoculate suspicious colonies into Hiss ' tube medium,
fermentation tube and milk. Also make indol test and try Widal
reaction.
c. Animal Inoculation. (Michigan method.)
1) Inoculate suspected water into bouillon tubes or flasks, and
incubate at 38° C.
2) Twenty- four to forty-eight hours later inoculate one cc. into
the peritoneal cavity of a white rat.
3) If animal recovers B. typhosus is not present. If animal dies
hold autopsy and isolate and study organism causing death.
Microspira comma.
a. If there be a reason to believe that the spirilla are very numer-
ous gelatin plate cultures can be made directly from the water, and
the suspicious colonies worked up.
360 MEDICAL BACTERIOLOGY
1}. Ordinarily the organisms are very sparse and large quantities
must be used. 100-1000 cc. are placed in flasks and \% of peptone
and 0.5% salt are added, the fluid made alkaline and incubated at
38° C. for 6-24 hours. Then gelatin plate cultures are made from
the upper layers and the suspicious colonies worked up as above.
Here and in typhoid the agglutination of the germ with great
dilution of a high potency serum is the crucial test.
Bacterium anthracis (Robert's Method.)
a. Heat suspected water to 80° for ten minutes to kill water
bacteria.
Z>. Make plates in agar and in gelatin and work up colonies.
c. Inoculate a guinea pig with several cubic centimeters of the
water.
REFERENCES. Horrocks and Prescott & Winslow.
EXEECISE 109. EXAMINATION OF MILK FOB PATHOGENIC
BACTEEIA.
Bacterium diphtheriae.
Where Bacterium diphtheriae is suspected in milk, make a con-
siderable number of streak cultures on Loeffler's blood serum and
incubate at 38° C. from 8 to 12 hours, stain and examine micro-
scopically.
Bacterium tuberculosis.
Hammond's method of examining milk for B. tuberculosis. See
Sputum, 101.
Animal Inoculation.
USE OF MAIL FOR TRANSMISSION OF BACTERIA.
Concerning the transmission of material containing bacteria in mails, see
Postal Guide, 1898, Euling No. 82, p. 901, part of which is as follows: "That
the order of the Postmaster-General of June, 1893, forbidding the use of
mails for the transmission of specimens of germs of cholera or other diseased
tissues, is hereby modified to this extent: Specimens of diseased tissue may
be admitted to the mails for transmission to United States, State or munic-
ipal laboratories only when inclosed in mailing packages constructed in
accordance with the specifications hereinafter enumerated. Upon the outside
of every package shall be written or printed the words: ' Specimen for Bac-
teriological examination/ No package containing diseased tissue shall be
delivered to any representative until a permit shall have first been issued by
the Postmaster-General, certifying that said institution has been found to
be entitled, in accordance with the requirements of this regulation, to receive
such specimens."
APPENDIX A
A KEY TO THE IDENTIFICATION OF THE COMMON PATHOGENIC
AND A FEW OF THE WELL KNOWN SAPEOPHYTIC BACTERIA
This key has been compiled from the works of Migula and Chester, the
latter of which contains a very complete key to practically all known bac-
teria. To this, as well as to Migula, Sternberg and Kolle and Wassermann
(for the pathogenic bacteria), the student is referred for detailed descriptions
of the various organisms.
Cells in their free condition globular (cocci).
A. Cells without flagella.
I. Division in only one direction of space forming chains (streptococci).
1. Grow on gelatin.
a) Do not liquefy gelatin.
1) No surface growth in gelatin stabs.
Sir. erysipelatos Fehleisen.
II. Division in two directions of space (micrococci).
1. Grow on gelatin.
a) Colonies white.
1) Do not liquefy gelatin.
M. Melitinsis Bruce.
2) Liquefy gelatin.
M. pyogenes var. albus ( Eosenbach ) L. & N.
b) Colonies yellow, and liquefy gelatin.
M. pyogenes var. awews(Rosenbach)L. & N,
2. Do not grow on gelatin.
M . gonorrhoeae (Baum) Fluegge.
M. Weichselbaumii (Trevisan).
III. Division in three directions of space (sarcinae).
1. Grow on gelatin.
a) Colonies white.
1) Do not liquefy gelatin.
Sar. tetragena (Gaffky) Mig.
b) Colonies yellow.
1) Do not liquefy gelatin.
Sar. lutea Fluegge.
Sar. ventriculi Goodsir.
2) Liquefy gelatin.
Sar. aurantiaca Fluegge.
Cells short or long, cylindrical, straight, without sheath, endospores present
or absent, non-motile (bacteria).
361
362 APPENDIX A.
A. Forms endospores.
I. Grow at room temperature.
1. Gelatin liquefied.
Bact. anthracis (Koch) Mig.
B. Without endospores.
I. Aerobic and facultative anaerobic.
1. Grow well on gelatin and do not liquefy it.
a) Gram's stain negative.
1) Gas generated in dextrose media.
i) Gas generated in lactose media.
Bact. aerogenes (Esch.) Mig.
Bact. capsulatum (Sternberg) Chester,
ii) Little or no gas in lactose media.
Bact. pneumonicum (Fried.) Mig.
2) No gas in dextrose media.
Bact. cholerae (Zopf) Kitt.
Bact. bovisepticum (Kruse) Mig.
(see also B. pestis)
b) Gram's stain positive.
1) Gas generated in dextrose media.
Bact. acidi-lactici Hueppe.
Bact. phosphor esc ens (Cohn) Fischer.
2) No gas in dextrose media.
Bact. rhinoscleromatis (Trevisan) Mig.
2. Gelatin liquefied slowly.
Bact. mallei (Loeffler) Mig.
Bact. rhusiopathiae (Kitt) Mig.
3. Do not grow well on gelatin at room temperature.
a) Stain with basic aniline dyes but are readily decolorized by
mineral acids when stained with carbol-fuchsin.
1) Grow well in bouillon at body temperature and stain by
Gram's method.
Bact. diphtheriae (Loeffler) Mig.
Bact. pseudodiphtheriticum (Loeffler) Mig.
2) Do not grow in bouillon or on ordinary media.
Bact. leprae (Hansen) Mig.
3) Growth very limited on ordinary media.
i) Gram's stain positive.
Bact. pneumoniae (Weichsel.) Mig.
ii) Gram's stain negative.
Bact. inftuenzae (Pfeiffer) L. & N.
b) Do not stain with aqueous solutions of basic aniline dyes and
not easily decolorized by acids.
Bact. tuberculosis (Koch) Mig.
Bact. tuberculosis var avium (Kruse) Mig.
II. Obligate anaerobic.
Bact. Welchii Mig.
Cells short or long, cylindrical, straight, without sheath, endospores present
or absent, motile, nagella distributed over whole body (bacilli).
APPENDIX A 363
A. Form endospores.
I. Aerobic or facultative anaerobic.
1. Potato cultures irregularly wrinkled.
B. vulgatus Trevisan.
4. Potato cultures smooth.
B. sub tills (Ehrenb.) Cohn.
I. Obligate anaerobes.
1. Eods not swollen at sporulation.
B. oedematis Zopf.
2. Eods spindle-shaped at sporulation.
B. Feseri (Trevisan) Chester.
B. botulinus v. Ermengen.
3. Eods clavate-capitae at sporulation.
B. tetani Nicolaier.
B. Spore formation not observed.
I. Aerobic or facultative anaerobic.
1. Gelatin colonies roundish not distinctly ameboid.
a) Gelatin not liquefied.
1) Gram's stain negative.
i) Generate gas in dextrose media.
* Coagulate milk.
§ Produce indol.
B. coll (Escherich) Mig.
§§ Do not produce indol.
B. enteritidis Gaertner.
** Do not coagulate milk.
B. Salmonii (Trevisan) Chester.
B. icteroides Sanerelli.
ii) Gas not generated in dextrose media.
B. typhosus Zopf.
B. dysenteriae Shiga.
B. peslis L. & N.
b) Liquefy gelatin.
1) Generate gas in dextrose media.
B. cloacae Jordan.
2) No gas generated in dextrose media, chromogenic, pig'
ment reddish.
B. prodigiosus (Ehrenb.) Fluegge.
2. Gelatin colonies ameboid or irregular.
a) Do not liquefy gelatin.
B. Zopfti (Kurth) Mig.
b) Liquefy gelatin.
B. vulgaris (Hauser) Mig.
Cells cylindrical, straight, without sheath, endospores known in only few
species. Actively motile, flagella attached to the poles (pseudomonas) .
A. Produce a greenish-bluish fluorescence in the culture media.
I. Gelatin liquefied.
364 APPENDIX A
1. Milk coagulated.
Ps. aeruginosa (Schroeter) Mig.
2. Milk not coagulated.
P$. ftuorescens (Fluegge) Mig.
II. Gelatin not liquefied.
1. Milk rendered alkaline.
Ps. syncyanea (Ehrenb.) Mig.
2. Milk reaction not changed.
Ps. putrida (Fluegge) Mig.
Cells cylindrical, more or less spirally curved, without endospores; actively
motile, flagella attached to the poles (microspira) .
A. Liquefy gelatin.
I. Produce indol in 24 hours.
1. Very pathogenic to pigeons..
Microspira Metschnikovi (Gamaleia) Mig.
Microspira $c7mt//A;t7/tewsis( Abbott) Chester.
2. Not distinctly pathogenic to pigeons.
Microspira comma (Koch) Schroeter.
II. Little or no indol in 24 hours.
Microspira Finklerii Schroeter
Cells in their ordinary form long branched filaments; cultures generally have
a mouldy appearance.
A. Gelatin liquefied.
Streptothrix bovis (Harz) Chester.
B. Gelatin not liquefied.
I. No distinct pigment on gelatin or agar.
Streptothrix farcinica Rossi-Doria.
II. Growths on gelatin or agar become reddish.
Streptothrix madurae Vincent.
Threads without distinct sheaths.
A. Without sulphur grains.
Lepothrix buccalis Miller.
B. With sulphur granules, motile, not attached.
Beggiato alba (Vaucher) Trevisan.
Threads with sheaths.
A. Without sulphur granules.
I. Without pseudodichotomous branching.
Crenothrix polyspora Cohn.
II. With pseudodichotomous branching.
1. Growths on gelatin whitish but gelatin stained brown.
Cladothrix dichotoma Cohn.
2. Gelatin not stained brown, colonies floccose— filamentous.
Cladothrix intrica Eussell.
B. With sulphur granules.
Thiothrix tenuissima Winogradsky.
APPENDIX B
Name of organism, source, habitat, etc.
REFERENCES.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
6. Agrar.
c. Gelatin.
d. Other media.
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet..
&. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILIT Y :
a. Character of movement..
&. Flagella stain
5. SPORES:
SPECIAL. CHARACTERS:
a. Capsules
&. Involution forms
c. Deposits or vacuoles..
d. Pleomorphism
365
CULTURE CHARACTERS
Eeaction of media (Fuller's scale) _j- or -
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48hoursat °C. I 6 days at *C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at CC.
Special Media: (Such as litmus milk and blood serum.)
APPENDIX B
367
Gelatin Stab: Grown 24 hours at °C.
O
48 hours at
6 days at ,C\
Agar Streak: Grown 24 hours at
48 hours at . .
6 days at °C.
Potato: Grown 24 hours at °C. Q|
n
h
48 hours at °C. 6 days at °C.
i
Bouillon: Grown 24 hours at °C.
2
\
48 hours at »C. 6 day sat...
368 PHYSIOLOGICAL CHARACTERS
1, RELATION TO TEMPERATURE: ,
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN :
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:.
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: COz: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OP NITRATES:
to nitrites to ammonia. . ,
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODPR:
11. PATHOGENESIS (or other special characters) :
APPENDIX B
369
Name of organism, source, habitat, etc.
REFEBENCES.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
i. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar.
e. Gelatin.
d. Other media.
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian-violet. .
b. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILIT Y :
a. Character of movement..
b. Flagella stain
5. SPORES:
SPECIAL CHARACTERS:
a. Capsules
b. Involution forms
c-. Deposits or vacuoles..
d. Pleomorphism ,
370 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) 4.
or
Gelatin plate: Grown 24 hours at °C.
(a) Surf ace Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at "C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at °C.
Special Media: (Such as litmus milk and blood serum.)
APPENDIX B
371
Gelatin Stab: Grown 24 hours at.
48 hours at
(lays at
Agar Streah: Grown 24 hours at
48 hours at
days at "C.
Potato : Grown 24 hours at °C.
\
48 hours at
6 days at..
Bouillon: Grown 24 hours at.
48 hours at °C.
6 days at..
372 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN :
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc. :— .
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm-
gas formula, H: COz: : :
&. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic. . .
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) : . ,
APPENDIX B
373
Name of organism, source, habitat, etc.
REFERENCES.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
ft. Agar.
c. Gelatin.
d. Other media..
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian-violet. .
ft. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY:
a. Character of movement.,
ft. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:
a. Capsules
ft. Involution forms
c. Deposits or vacuoles..
d. Pleomorphism
374 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -j_
Gelatin plate: Grown 24 hours at °C.
[a) Surface Colonies (b) Deep Colonies,
Sketches,
48 hours at , .......... °C,
;
6 days at.,.,...,,...,,. *C.
Agar plate; Grown 24 hours at.
(a) Surface Colonies,
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at
Special Media: (Such as litmus milk and blood serum.)
APPENDIX B
375
Gelatin Stab: Grown 24 hours at °C.
Agar Streak: Grown 24 hours at ,
48 hours at °C
I
a!/
48 hours at . .
6 days at °C.
Potato: Grown 24 hours at
48 hours at
6 days at.
Bouillon: Grown 24 hours at °C.
48 hours at "C.
6daysat "C.
376 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: « t
optimum °C. ; limits to °C. ;
thermal death-point °C. ; time of exposure minutes
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION;
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture :
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent.. 48 hours percent.
72 hours per cent .hours per cent,
reaction in open arm
gas formula, H: COa: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:... .
litmus milk
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) ;
APPENDIX B
377
Name of organism, source, habitat, etc.
REFERENCES.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agrar-
c. Gelatin.
d. Other media..
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet. .
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
&. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:....
a. Capsules
6. Involution f orms
c. Deposits or vacuoles.
d. Pleomorphism .
378 CULTURE CHARACTERS
Eeaction of media (Fuller's scale) -f_ or —
Gelatin plate: Grown 24 hours at °C.
Surface Colonies. (b) Deep Colonies.
Sketches.
48hoursat ......................... °C. 6 days at
«C.
Agar plate; Grown 24 hours at °C. I Sketches.
(a) Surface Colonies.
(b) Deep Colonies.
48 hours at °C.
6 days at *C.
Special Media: (Such as litmus milk and blood serum/
APPENDIX B
379
Gelatin Stab: Grown 24 hours at °C ^j <
.
9
i
\ /
hours at °C
u
c
*-> • \
OS ,/
2
!^
Q
O
Z
2
3
o
A
A
U
48 hours at °C 6 days at
0
c.
Potato : Grown 24 hours at °C
;A
.a
hours at °(
/\
/ \
/ \
w
o
c
Bouillon: Grown 24 hours at °C r\
.
*
X
A
O
I
V J
48 hours at °C. 6 days at. . .
0
c.
380 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum °C. ; limits to °C.;
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:...
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent., 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H: CCb: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OP NITRATES:
to nitrites to ammonia..,
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION: . .
proteolytic
digestion of gelatin digestion of casein.
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
APPENDIX B
381
Name of organism, source, habitat, etc.
REFERENCES.
MORPHOLOGICAL CHARACTERS :
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
&. Agrar.
c. Gelatin.
d. Other media.,
2. SIZE:
3. STAINING POWERS :
a. Aqueous gentian-violet..
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILJTY :
a. Character of movement..
&. Flagella stain
5. SPORES:
6. SPECIAL CHARACTERS:
a. Capsules
6. Involution forms
c. Deposits or vacuoles.
d. Pleomorphism
382 CULTURE CHARACTERS
Eeactiou i- media (Fuller's scale) -{- or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies.
(b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at »C.
Agar plate: Grown 24 hours at °C.
(a) Surf ace Colonies. (b) Deep Colonies.
Sketches.
48 Hours at °C.
6 days at
Special Media: (Such as litmus milk and blood serum.)
APPENDIX B
383
Gelatin Stab: Grown 24 hours at CC.
48 hours at
6 days at <>C.
Streak: Grown 24 hours at °C.
O
48 hours at
6 days at °C.
Potato : Grown 24 hours at °C.
Bouillon: Grown 24 hours at.
•C.
48 hours at °C
6 days at °C
48 hours at °C.
6 days at.
384 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE: , ,
optimum °C. ; limits to °£. ;
thermal death-point °C.; time of exposure minutes;
medium in which exposure is made
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
6. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2) Fermentation tube, growth in open arm closed arm
rate of development: 24 hours per cent., 48 hours percent.
72 hours per cent., hours per cent.
reaction in open arm
gas formula, H: COz: : :
6. lactose c. saccharose
6. ACID OR ALKALI PRODUCTION:
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia.. .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :.
APPENDIX B
385
Name of organism, source, habitat, etc.
REFERENCES.
MORPHOLOGICAL CHARACTERS:
SKETCHES.
1. FORM AND ARRANGEMENT:
a. Bouillon
b. Agar.
c. Gelatin.
d. Other media..
2. SIZE:
3. STAINING POWERS:
a. Aqueous gentian- violet..
6. Loeffler's methylen-blue.
c. Gram's stain
d. Special stains
4. MOTILITY :
a. Character of movement..
6. Flagella stain
. SPORES:.
6. SPECIAL CHARACTERS:....
a. Capsules '.
6. Involution forms
c. Deposits or vacuoles.
d. Pleomorphism
386 CULTURE CHARACTERS
Keaction of media (Fuller's scale) _-
or —
Gelatin plate: Grown 24 hours at °C.
(a) Surface Colonies. (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at «C.
Agar plate: Grown 24 hours at °C.
(a) Surface Colonies. : (b) Deep Colonies.
Sketches.
48 hours at °C.
6 days at.
Special Media: (Such as litmus milk and blood serum.)
APPENDIX B
387
Gelatin Stab: Grown 24 hours at °C.
48 hours at °C.
6 days at
Agar Streak: Grown 24 hours at °C.
o o
/\
/
I ;
48 hours at °C. 6 days at CC.
Potato: Grown 24 hours at °C. >j
/ \
« / \ a
i i I
y
48 hours at °C. 6 days at °C.
Bouillon: Grown 24 hours at.
48 hours at °C.
6 days at °C.
388 PHYSIOLOGICAL CHARACTERS
1. RELATION TO TEMPERATURE:
optimum ° C. ; limits to °C.:
thermal death-point °C. ; time of exposure minutes;
medium in which exposure is made »
2. RELATION TO FREE OXYGEN:
3. RELATION TO OTHER AGENTS, SUCH AS
desiccation, light, disinfectants, etc.:—.
4. PIGMENT PRODUCTION:
5. GAS PRODUCTION IN SUGAR MEDIA:
a. dextrose (1) Shake culture:
(2* Fermentation tube, growth in open arm closed arm
rate of development: 24 hours percent.. 48 hours percent.
72 hours per cent hours per cent.
reaction in open arm
gas formula, H : COs : : :
v. lactose c. saccharose •
6. ACID OR ALKALI PRODUCTION:...
litmus milk.
7. REDUCTION OF NITRATES:
to nitrites to ammonia. . .
8. INDOL PRODUCTION
48 hours days .
9. ENZYME PRODUCTION:
proteolytic
digestion of gelatin digestion of casein .
diastatic
10. CHARACTERISTIC ODOR:
11. PATHOGENESIS (or other special characters) :
APPENDIX C
TABLES
COMPARISON OF THERMOMETERS.
(From GQiild's Dictionary of Medicine)
a
I
I
ID
•<
H
«
A
1
H
0
p
«
I
H
o
p
212
100
80
122
50
40
32
0
0
210
98.9
79.1
120
48.9
39.1
30
-1.1
-0.9
208
97.8
78.2
118
47.8
38.2
28
-2.2
-1.8
206
96.7
77.3
116
46.7
37.3
26
-3.3
-27
204
95.6
76.4
114
45.6
36 4
24
-4.4
-3.6
202
94.4
75.6
112
44.4
35.6
22
-5.6
-4.4
200
93.3
74.7
110
43.3
34.7
20
-6 7
-5.3
198
92.2
73.8
108
42.2
33.8
18
-7.8
-6.2
196
91.1
72.9
106
41.1
32.9
16
-8 9
-7.1
194
90
72
104
40
32
14
-10
-8
192
88.9
71.1
102
38.9
31.1
12
-11 1
-8.9
190
87.8
70.2
100
37 8
30.2
10
-12.2
-9.8
188
86.7
69.3
98
36.7
29.3
8
-13.3
-10.7
186
85.6
68.4
96
35.6
28.4
6
-14.4
-11.6
184
84.4
67.6
94
34.4
27.6
4
-15.6
-12.4
182
83.3
66.7
92
33.3
26.7
2
-16.7
-13.3
180
82.2
65.8
90
32.2
25.8
0
-17.8
-14.2
178
81.1
64.9
88
31.1
24.9
-2
-18.9
-15.1
176
80
64
86
30
24
-4
-20
-16
174
78.9
63.1
84
28.9
23.1
-6
-21.1
-16.9
172
77.8
62.2
82
27.8
22.2
-8
-22.2
-17.8
170
76.7
61.3
80
26.7
21.3
-10
-23.3
-18.7
168
75.6
60.4
78
25.6
20.4
-12
-24.4
-19.6
166
74.4
59.5
76
24.4
19.6
-14
-25 6
-20.4
164
73.3
58.7
74
23.3
18.7
-16
-26.7
-21.3
162
72.2
57 8
72
22.2
17.8
-18
-27.8
-22.2
160
71.1
56.9
70
21.1
16.9
-20
-28.9
-23.1
158
70
56
68
20
15
-22
-30
-24
156
68.9
55.1
66
18.9
15.1
-24
-31 1
-24.9
154
67.8
54.1
64
17.8
14.2
-26
-32.2
-25.8
152
66.7
53.3
62
16.7
13.3
-28
—33 . 3
-26.7
150
65.6
52.4
60
15.6
12.4
-30
-34.4
-27.6
148
64.4
51.6
58
14.4
11.6
-32
-35.6
-28.4
146
63.3
50.7
56
13.3
10.7
-34
-36.7
-29.3
144
- 62.2
49.8
54
12.2
9.8
-36
-37.8
-30.2
142
61.1
48.9
52
11.1
8.9
-38
-38.9
-31.1
140
60
48
50
10
8
-40
-40
-32
138
58.9
47.1
48
8.9
7.1
-42
-41.1
-32.9
136
57.8
46.2
46
7.8
6.2
-44
-42.2
-33.8
134
56.7
45.3
44
6.7
5.3
-46
-43.3
-34.7
132
55.6
44.4
42
5.6
4.4
-48
-44.4
-35.6
180
54.4
43.6
40
4.4
3.6
-50
-45.6
-36.4
128
53.3
42.7
38
3.3
2.7
-52
-46.7
-37.3
126
52.2
41.8
36
2.2
1.8
-54
-47.8
-38.2
124
51.1
40.9
34
1.1
0.9
-56
-48.9
-39.1
To change Centigrade to Fahrenheit: (C X f ) + 32 = F.
For example, to find the equivalent of 10° Centigrade, C = 10° (10° X f)
+ 32 = 50° F.
To change Fahrenheit to Centigrade: (F. — 32°) X f = C.
For example, to reduce 50° F. to Centigrade, F. = 50° and (50° — 32°)
X f = 10° C. or — 40° F. to Centigrade, F. = — 40° (— 40° — 32°) = — 72°,
whence — 72° X f = — 40° C.
389
390 APPENDIX C
COMPARATIVE LIST OF METRIC AND ENGLISH SYSTEMS.
Metre = 100 centimetres, 1000 millimetres, = 39.3704 inches.
Millimetre = 1000 microns, -£% inch, approximately.
Inch = 25.399772 mm. (25.4 approximately).
Litre = 1000 millilitres or 1000 cc., 1 quart (approximately).
Cubic Centimetre = y^V^ of a litre.
Fluid ounce (8 fluid drachms) = 29.578 cc., (30 cc., approximately).
Gram = 15.432 grains.
Kilogram = 2.204 avoirdupois pounds (2^ pounds, approximately).
Ounce, avoirdupois, =(437^ grains) =28. 349 grams ) 30 grams, ap*
Ounce, Troy or apothecaries, =(480 grains) =31. 103 grams, ) proximately.
INDEX
ABBE, condenser, 34.
Absorbent cotton, 14.
Acetic acid, decolorizing action, 60.
Acid
acetic, 60.
alcohol, 312, 316.
carbolic, use in detecting enzymes, 80.
in Pariette's solution, 338.
production, 76.
pyrogallic, 272.
sulphanilic, 76.
Acids, determination of, 76.
Acidity of Media, 8.
Actinomyces bovis, 263.
Aerobes, 72, 74.
pathogenic, 138.
Agar culture medium, 16.
dextrose, 64.
filtering of, 16.
hanging-drop culture, 44.
lactose, 64.
melting point of, 56.
plate cultures, 56.
preparation of, 16.
slopes, 16.
sterilization of, 16.
Air analysis. 126.
pump, 14.
Alcohol, as a decolorizing agent, 60.
Alcoholic solutions of dyes. 28.
Alkalies, detection of in cultures,. 76.
Alkalinity of media, 8.
Ameba coli. 344. 352.
Ammonia in cultures, 78.
Anaerobes, 72, 74.
pathogenic, 92, 271.
Wright's Method of Cultivating, 272.
Analysis of Air, 126.
of gas, 74.
of milk, 132.
of soil, 132.
of wa.er, 128,
Anilin dyes, 28.
water, 30.
Animal autopsy, 307.
cage, 300.
care of inoculated, 300.
experiment, blank for, 308.
inoculation, 294.
Anthrax bacillus, 1^7.
group, 91, 167.
Antiseptic action, 72.
Antiseptics, testing of, 134.
Apparatus for cooling plates, 54.
for tubing media, 10.
Arnold sterilizer. 10.
Arrangement of bacteria, 91.
Asiatic cholera germ, 251.
Asparagin, 46.
Aspergillus, 50.
Aspirator, 126.
Autoclave, 12.
Autopsy, animal, 302.
cultures at, 304.
human, 356.
instruments, 300.
T5ACILLTJS, 89.
-15 acidi lactici, 109.
aerogenes, 175.
capsulatus, 273.
amylobacter, 46.
Bacillus
anthracis, 167.
symptomatici, 277-
botulinus, 285.
bovisepticus, 187.
capsulatum, 179.
carbonis, 277.
cnolerae gallinarum, 183.
suum, 227.
coli, biology of, 219.
effect of chemicals on, 134.
desiccation. 70.
reaction of medium, 6&
for removing sugars, 64.
Gram's stain for, 62.
in water, 130.
motility, 40, 50.
plate cultures, 56, 58.
production of acids, 76.
gases, 74.
hydrogen sulphide, 78.
indol, 78.
thermal death point, 68.
diphtheria?, 199.
dysenteriae, 239, 344.
edematis, 281, 314, 318, 352.
maiigni 381.
enteritidis, 223.
Feseri. 277, 314, 318.
icteroides, 231.
influenzas, 211.
mallei, 191.
mesentericus vulgatus, 95.
mycoides, 40, 42.
of blue-green pus, 247.
of bubonic plague, 243.
of chicken cholera, 183.
of glanders 191.
of hemorrhagic septicemia, 187.
of Japanese dysentery, 239.
of malignant edema, 281.
of swine plague. 182.
of swine erysipelas, 195.
of symptomatic anthrax, 277.
pestis, 243, 306. 330, 334, 352.
bubonicae, 243.
pneumonicae, 171.
prodigiosus, 74, 80, 103, 108.
proteus, 113.
pyocyaneus, 247.
Salmonii, 227, 306, 318, 334.
septicemiae hemorrhagicse, 183.
subtilis, 24. 25, 40, 46, 48. 50, 54, 58,
62, 66, 70, 72, 76, 80, 99.
suipestifer, 227.
typhi abdominalis, 235.
typhosus. 50, 70, 235, 318, 336, 338,
346. 3.18.
tetani. 46. 289, 306, 314, 352.
tuberculosis, see Bact. tuberculosis.
vulgaris, 113.
vulgatus. 95.
Zopfii, 117.
Bacteria
arrangement in groups, 91.
capsule stain for, 50, 314.
cell grouning of, 42.
classification, 89.
colonies of, 52.
compared with yeasts and moulds, 50.
decolorizing, 60.
determining size, 38.
391
392
INDEX
Bacteria
drawing, 36.
effect of chemicals on, 72.
of desiccation on, 70.
of direct sunlight on, 72.
of moist and dry heat on, 70.
flagella stain, 48.
formation of ammonia by, 78.
of enzymes by. 80.
of gas by, 74.
of indol by, 78.
of sulphuretted hydrogen by, 78.
involution forms, 46.
metachromatic granules, 50.
movement of, 40.
non-pathogenic, 93.
pathogenic, 138, 271.
reduction of nitrates by, 76.
relation to oxygen, 72.
staining, 30.
in tissue, 312.
spores of. 46.
thermal death point of, 68.
Bacteriacese, 89.
Bacterium, 89.
acidi-lactici, 109.
jrogenes, 175.
'anthracis, 46, 167, 304, 314, 330, 360.
bovisepticum, 187.
capsulatum, 50, 179.
cholerse, 183, 316.
cholerse gallinarum, 183.
suum, 227.
coli commune, 219.
diphtherise, 46, 50, 199, 3C6, 314, 318,
360.
diagnosis, 320.
erysipelatos suis, 195.
influenzse, 211.
lactis serogenes, 175.
- leprse, 314, 316, 350.
mallei, 191, 306, 316, 332, 350.
pestis, 243.
phosphorescens, 121.
pneumonise, 50, 207, 304, 314, 328,
332, 350.
pneumonicum, 50. 171, 304, 314.
pseudodiphthereticum, 203, 322.
rhusiopathise, 195, 314.
suicida, 183.
tuberculosis, 213, 304, 314, 316, 326,
334, 344, 346, 350, 360.
Gabbett's stain, 62.
in sputum, 326.
in tissue, 316.
in urine, 346.
Welchii, 273, 314, 318, 352.
Zopfli, 117.
Bacteriological analysis, 126.
diagnosis, 320.
Beef extract, 6.
Beggiatoa, 90.
Beggiatoacese, 90.
Bismarck brown, 28.
Black-leg bacillus, 277.
Blood, agar, 212.
examination of, 330.
serum, 138.
Boehmer's hematoxylin, 312.
Bolton's potato tubes, 18.
Bouillon cultures, 22, 87.
dextrose, 64.
lactose, 64.
preparation of, 6.
saccharose, 64.
sugar-free, 64.
Brownian movement, 40.
Buccal secretion, 320.
Buchner's method of growing anaerobes,
272.
uunge's flagella stain, 48.
PAGE for animals, 300.
^ Calcium chloride, 6.
Canada balsam, 32.
Capsule stain, 50, 314.
Carbol-fuchsin, 28, 34.
Carbolic acid, 28, 72, 80.
Care of culture media, 18.
Cell grouping, 42.
Celloidin sections, 310.
Centrifuge, 32#.
Chain coccus, 141.
Chemicals, antiseptic action of, 134.
disinfecting action of, 136.
effect on bacteria. 72.
Chlamydobacteriaceae, 90.
Cholera group, 92, 251.
red reaction, 78, 344.
Chromogenic class, 91, 103.
Cladothrix, 90.
Cleaning glassware, 2.
slides and cover-glasses, 26.
solution, 28.
Coccaceae, 89.
Coccus of Malta fever, 153.
Collodion sacs, 296.
Colon bacillus, see li. coli.
group, 92, 219.
Colonies, 52, 83.
color of, 85.
counting, 126.
miscroscopic structure, 84.
surface elevation, 84.
types of, 83.
Color of colonies. 85.
production, variation in, 80.
Coloring matter, separation of, 108.
Concentration of media, 66.
Condensed milk, 66.
Condenser, Abbe, 34.
Cooling apparatus, 54.
Cornet forceps, 32.
Corrosive sublimate, IX.
Cotton, 4.
absorbent for filtering, 14.
Counting apparatus, 126.
Cover-glass preparations, 30.
Cover-glasses, cleaning of, 26.
Crenothrix, 90.
Culture characters of bacteria, 83.
media, 6, 10, 64, 138.
of anaerobes, 271.
Cultures, agar, 22.
plate, 52.
roll, 56.
bouillon, 84.
gelatin, stab. 22.
plate, 52.
hanging-drop, 44.
incubation of, 24.
potato, 24
set of, 93.
stab, 22.
streak, 24.
test-tube, 22.
"DECOLORIZING agents, so.
•*-' Desiccation, effect on bacteria, 70
Dextrose agar, 64.
bouillon, 64.
free bouillon, 64.
gelatin, 62.
Diagnosis, methods of, 320.
of rabies. 354.
Diaphragm, iris, 36.
Diphtheria bacillus, see B. diphtheria
group, 92, 199.
outfit, 320.
Diplococcus gonorrhoeae, 157.
group, 91, 157.
intracellularis meningitldls, 159.
pneumonise, 257.
Disinfectants, 72, 136=
Drawing bacteria, 36.
INDEX
393
Drigalski & Conradi's medium, 342.
Dry air sterilizer, 4.
Dunham's clearing solution, 312.
peptone solution, 64.
Dyes, anilin, 28.
EGG, use in clearing media, 14.
Ehrlich's anilin oil gentian violet, 28.
Embedding tissue, 310.
Emphysema group, 92, 273.
Endospores, study of, 46.
Enzymes, 80.
Eosin, 312.
Ernst's stain, 50.
Erysipelas group, 91, 141.
Esmarch's roll cultures, 56.
Examination of blood, 330.
of buccal secretion, 320.
of feces, 338.
of sputum. 326.
of transudates and exudates, 346.
of urine, 346.
Extract of beef, 6.
Exudates, examination of, 346.
Eyepieces, see ocular.
FECAL bacteria, 130.
Feces, examination of, 338.
Fermentation tube, 74. \
Filling tubes and flasks, "
Filter, folded, 8.
pump, 14.
Filtering gelatin, 14.
agar, 16.
Flagella stain, 48.
Folder filter, 8.
Forceps, Cornet, 32.
cover-glass, 32.
Stewart, 32.
Form types, study of, 40.
Fraenkel's soil borer, 132.
Fnedlander's bacillus, 171.
group, 91, 171.
Frost's gasometer, 74.
Frozen sections, 310.
Fuchsin, 28,
p ABBETT'S methylen blue, 30.
^ tubercle stain, 62.
Gas analysis, 74.
formula, 74.
Gasometer, 74.
Gelatin, dextrose, 64.
filtering of, 14.
melting point of, 24.
plate cultures, 52.
preparation of, 14.
reaction of, 14.
sterilization of, 14.
Gentian violet, 28.
Glanders bacillus, 191.
group, 91, 191.
£}lass slides, cleaning of, 26.
hollow ground, 38.
Jumblers, 24.
Glassware, cleaning of, 2.
sterilization of, 4.
Golden pus coccus, 149.
Gonococcus. 157.
Gonorrhceal pus, 348.
Gram's iodine solution, 30.
stain, 60.
list of organisms taking this stain,
314.
Guinea pig, diagram, 307.
inoculation of, 294.
Groups, arangement of bacteria in, 91.
HAMMOND'S method of concentrating
tubercle bacilli in milk and sputum,
328.
Hanging-drop culture, 44.
preparation, 38, 44.
Hauser's spore stain, 48.
Hay bacillus, 24.
Heat, effect of on bacteria, 68.
Hematoxylin stain, 312.
Hiss' culture medium, 340.
Hog cholera bacillus, 227.
group, 92, 227.
Hot air sterilizer, 4.
Hydrochloric acid, 2, 8.
Hydrogen generator, 271.
TMMERSION lens, 34.
A Impression preparations, 42.
Incubation of cultures, 24.
Incubator, 24.
Indol test, 78, 130.
Influenza bacillus, 211, 328.
group, 92, 211.
Inoculated animals, care of, 300.
Inoculation of animals, 294.
into ear, 298.
into eye, 300.
intraperitoneal, 294.
intrapleural, 300.
intravenous, 298.
lymphathic, 300.
subcutaneous, 294.
subdural, 3o4.
Instruments, sterilization of, 300.
Involution forms, 46.
Iodine solution, 30, 314.
Iris diaphragm, 34.
TEFFER'S counting apparatus, modified,
J Plate II, p. 137.
TTEY to identification of bacteria, 361.
AV Kipp hydrogen generator, 271.
Klebs-Loeffler's bacillus, 199.
Koch's method of air analysis, 126.
T ABELS, 20.
*-* Lactic acid bacillus, 109.
Lactose agar, 64.
litmus agar, 76.
Lens paper, 36.
Litmus milk, 66.
solution, 64.
Loeffler's blood serum, 138.
methylen blue, 28.
stain for sections, 312.
Loop, platinum, 20.
IVfACCONKEY'S medium, 340.
ltj- Malaria, 326.
Mallory's stain for ameba coli, 334.
Malta fever group, 91, 153.
Media, care of, 12, 18.
examination of, 12.
labeling, 20.
preparation, 6-18, 64, 94, 138.
reaction of, 8.
sterilization of, 10.
Measuring bacteria, 36.
Mesophilic bacteria, 68.
Metachromatic granules, stain for, 50.
Methylen blue, alkaline, 28.
Gabbett's 30.
Loeffler's, 28.
Mice, white, 294.
Micrococcus, 89.
gonorrhceae, 157. 314, 346, 348.
melitensis, 153.
pyogenes, var. albus, 145, 314, (see
also pus micrococci).
var. aureus, 149, 314 (see also pus
micrococci) .
Weichselbaumii, 159, 350.
Micrometer, ocular, 36.
stage, 36.
Micron, 38.
Microspira, 89.
comma, 251, 342, 358,
394
INDEX
Microspira
Metschnikovi; 40, 255.
Schulykilliensis, 259.
Microscope, 34.
Microscopic study of plate cultures, 58.
of bacteria, 40.
Migula's classification, 89.
Milk anaylsis, 132, 360.
cultures, 87.
litmus, 66.
pasteurization of, 132.
Molecular movement, 40.
Monilia Candida, 324.
Monas prodigiosa, 103.
Morphology, 2, 82.
Motility of bacteria, 40.
Moulds, morphology of, 50.
Mucor, 50.
•V"EEDLE, platinum, 20.
f1 Neisser's diphtheria stain, 322.
Nessler's reagent, 78.
Neutralization of media, 6.
Nitrate solution, 64.
Nitric acid, decolorizing, 60.
Nitrites, detection of. 76.
Nitroso-indol (cholera red), 78.
Non-pathogenic bacteria, 93.
Normal solutions, 8.
Novy's anaerobic jars, 271.
QBJECTIVE, oil-immersion, 34.
^ Ocular micrometer, 36.
Ocular, 34.
Oedema group, 92, 277.
Oil-immersion ojective, 34.
Oospora bovis, 263.
Oxalic acid, 344.
Oxygen, relation of growth to, 72.
PARAFFIN, sealing tubes with, 20.
x sections, 310.
Parietti's solution, 338.
Pasteurization of milk, 132.
Pathogenic aerobes, 91, 138.
anaerobes, 271.
bacteria, reaction of media for, 8.
best temperature for, 24.
in water and milk supplies, 358.
Penicillium, 50.
Peptone, 6.
Petri dish cultures. 52.
Petri-Sedgwick's air analysis, 126,
Pfeiffer's capsule bacillus, 179.
stain, 318.
Phenolphthalein, 8.
Phosphorescent class, 91, 121.
Photobacterium phosphorescens, 121.
Phragmidiothrix, 90.
Physiology of bacteria, 64.
Physiological characters of bacteria, 88.
Pigeons, inoculation of, 294.
Pigments, separation of. 107.
variety of, 108.
Planococcus, 89.
Planosarcina, 89.
Plasmodium, malariae, 334.
Plasmolysis, 83.
Plate cultures, agar, 56.
gelatin, 52.
method of pouring, 54.
study of, 58.
Platinum needles. 20.
Plugging flasks and tubes, 4.
Pneumobacillus, 171.
Pneumonia group, 92, 207.
Post-mortem examination, 302.
Potassium hydrate. 28.
Potassium iodide, 30.
Potato bacillus, 95.
Potatoes, preparation of, 18.
Preparation of agar, 14.
of bouillon, 6.
of cover-glasses, 30.
Preparation of gelatin, 14.
of hanging-drop, 38.
of potatoes, 18.
of staining solutions, 28.
of water blanks, 18.
Proteus vulgaris, 113.
Zenckeri, 117.
Pseudodiphtheria bacillus, 203.
Pseudomonas, 89.
ffiruginosa, 80, 247, 314, 352.
fluorescens, 40.
group, 92, 247.
violacea. 68.
Psychrophilic bacteria, 68.
Piis micrococci, 314. 324, 332, 346, 348.
Pyrogallic acid, 272.
-RABBITS, 294.
**> Rabies, diagnosis of, 354.
Rats, white. 294.
Ravenel's method of making agar, 16.
on diagnosis of rabies. 354.
Ray fungus. 263.
Reaction of media, 8. 66.
Ribbert's method of concentrating the
tubercle bacteria in sputum, 326.
Rice cooker, 6.
Roll cultures, 56.
Rubber dam for sealing tubes. 20.
Russell's water sampler, 128.
OACCHAROMYCES cerevisise, 50.
a Salt, 6.
solution, physiological, 18.
Saprogenic class. 91, 113.
Saprophilic class, 91, 95.
Sarcina, 89.
group. 91, 163.
lutea, 40.
tetragena, 163. 304, 314.
Sections, 310, 312.
Sedgwick's method of air analysis, 126.
Shake culture, 74.
Slides, cleaning of, 26.
Smegma bacillus, 346.
Spirillacese, 89.
Spirillum, 90.
cholerse Asiaticse, 251.
rubrum, 4.
Spirochaeta. 89.
Obermeieri, 332, 346.
Spirosoma, 89.
Spore stain, 48.
Spores, study of, 46.
Sputum, centrifugalizing, 328.
examination of for bacteria, 326.
stain of for tubercle bacteria, 62, 326.
Soapstone for cooling plates, 54.
Sodium chloride, 6.
hydrate, 8.
nitrate solution, 64.
nitrite, 78.
Soil analysis, 132.
Stage micrometer. 36.
Stain, ahilin, 28.
bottles. 30.
Bunge's for. flagella, 48.
Ernst's, 50.
Gabbett's. 62.
Gram's, 60.
Hauser's, 48.
Loeffler's, 312.
solutions, 28.
Weigert's, 314.
Ziehl's, 326.
Stab cultures, 22, 85.
Staphylococcus, epidermidis albus, 145.
pyogenes albus, 145.
pyogenes aureus, 149.
Steam pressure, 12.
sterilizer, 10.
Sterilization, discontinuous, 10.
of glassware, 4.
INDEX
395
Sterilization of instruments, 300.
of media, 10.
Sterilizer, hot air, 4.
Sternberg's bulbs, 330.
Streak cultures. 24, 86.
Streptococcus, 89.
erysipelatos, 141, 304, 314, 348.
lanceolatus, 307.
pyogenes, 141.
Streptothrix, 90.
actinomyces, 263.
bovis, 263, 314, 318, 330, 352.
group, 92, 263.
Madurse. 267.
Sugar meuia, sterilization of, 12.
Sulphanilic acid, 76.
Sulphuric acid, 30.
Sulphuretted hydrogen, 78.
Sunlight, effect on bacteria, 72,
Swine plague group. 91, 183.
Swine erysipelas, 195.
Syringe, sterilization of, 300.
use of, 294.
^TAXONOMY, 82.
-1- Temperature, effect on growth, 68.
Testing antiseptic action, 134.
disinfecting action, 136.
Test-tube, cleaning of, 2.
cultures, 22.
igar, 24.
)lood
blood serum, 24.
gelatin, 22.
potato. 24.
study of, 26.
staining of, 30.
Tetanus group, 92, 289.
Thermal death point, 68.
Thermo-regulator, 12, 26.
Thermostat, 24.
Thionin, 344.
Thiothrix, 90.
Thrush, organism of, 324.
Tin foil, for sealing tubes, 20.
Tissue embedding, 310.
fixing, 310.
handling sections, 310.
preparation of for examination, 310.
sectioning, 310.
Tissue, staining of bacteria in, 312
Titration of media, 6. '
Transudates, examination of, 346.
Tubercle bacillus, 213.
group, 92, 213.
stain. 6^, 326.
Tyndallization, 10.
Typhoid bacillus, 235.
group, 92, 235.
•JJRINE, examination of, 346.
VIBRIO cholerae, 251.
v Metschnikovi, 255.
Schuylkilliensis, 259.
subtilis, 99.
Van Gehucten & Xeli&' method of diag-
nosing rabies, 354.
V\rATER analysis, 128, 358.
reaction of media for, 8.
bath, 56.
blanks, 18.
Weigert's iodine solution, 314.
stain, 314.
Welch's capsule stain, 50.
Wertheim's medium for the gonococcus,
158. 3*8.
Wesbrook's animal cage, 300.
hanging-drop culture, 44.
White mice. ^94.
rats. 294.
staphylococcus. 145.
Widal reaction. 334.
Wright's method of cultivating anaerobes,
Wurtz' lactose litmus agar, 76.
bacillus, 203.
YEASTS> morphology of, 50.
carbol-fuchsin, 28.
Ziehl-Neelsen's stain. 326.
Zymogenic class, 91, 109.
Printed in the United States of America.
XC 08529
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